Textile dye degrading laccase from Pseudomonas desmolyticum NCIM 2112

Enzyme and Microbial Technology 44 (2009) 65–71

Contents lists available at ScienceDirect

Enzyme and Microbial Technology journal homepage: www.elsevier.com/locate/emt

Textile dye degrading laccase from Pseudomonas desmolyticum NCIM 2112 Satish Kalme a,1 , Sheetal Jadhav b , Mital Jadhav b , Sanjay Govindwar a,∗ a b

Department of Biochemistry, Shivaji University, Kolhapur 416004, India Department of Microbiology, Shivaji University, Kolhapur 416004, India

a r t i c l e

i n f o

Article history: Received 23 April 2008 Received in revised form 3 October 2008 Accepted 4 October 2008 Keywords: Laccase Degradation Pseudomonas desmolyticum Textile dye FTIR

a b s t r a c t A laccase requiring optimum temperature 60 ◦ C, pH 4.0 for the activity and having apparent molecular weight 43,000 Da was purified from Pseudomonas desmolyticum NCIM 2112 by three steps, including heating, anion exchange, and molecular sieve chromatography. The purification fold and yield of laccase obtained through Biogel P100 were 45.75 and 19%, respectively. Staining of native gel with L-dopa showed dark brown color band indicating the presence of laccase. In relation to hydroquinone, the substrate specificity of laccase was in the following order: DAB > o-tolidine > ABTS > L-dopa. The absence of monophenolase activity in eluted fractions conformed that the purified protein is laccase. This laccase showed substrate dependent optimum pH character. Effect of inhibitor and metal ion on enzyme activity was analyzed. UV–vis analysis showed the decolorization of Direct Blue-6, Green HE4B and Red HE7B in the presence of laccase. The FTIR spectral comparison between the control dye sample and the metabolites extracted after decolorization by purified laccase have confirmed degradation of these dyes. This study contributes for the structural requirement of a dye to be degradable by P. desmolyticum laccase and is important in order to optimize potential bioremediation systems for industrial textile process water treatment. © 2008 Elsevier Inc. All rights reserved.

1. Introduction The biodegradation ability of the bacteria is assumed to be associated with the production of lignolytic enzymes such as lignin peroxidase [1] and laccase [2]. Laccase (EC 1.10.3.2, benzenediol: oxygen oxidoreductase) is a multicopper oxidase, widely distributed among plants, fungi [3], and bacteria [4]. It catalyzes the oxidation of a broad range of organic and inorganic substrates, including diphenols, polyphenols, diamines, aromatic amines, and ascorbate by a one-electron transfer mechanism [5]. There are extensive studies focused on laccase from fungi like Daedalea quercina [6], Sclerotium rolfsii [7], Ganoderma lucidum [8], Trametes trogii [9], and Pycnoporus sanguineus [10]. Laccases can be applied extensively in many fields, including waste detoxification and textile dye transformation due to their low substrate specificity [2]. Although some bacterial laccases have been characterized

Abbreviations: DAB, 3-3 -diaminobenzidine tetrahydrochloride; ABTS, 2,2 azinobis (3-ethylbenzthiazoline-6-sulfonate; DB6, Direct Blue-6; GHE4B, Green HE4B; RHE7B, Red HE7B. ∗ Corresponding author. Tel.: +91 231 2609152; fax: +91 231 2691533. E-mail address: spg [email protected] (S. Govindwar). 1 Present address: Department of Chemistry, Protein Biotechnology Lab, Hanyang University, Seoul 133791, Republic of Korea. 0141-0229/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2008.10.005

[11,12], little information is available concerning their substrate specificities towards dye decolorization. Large amounts of chemically different dyes are used for textile dyeing and a significant proportion of these dyes enter the environment as waste water. Not all these dyes could be degraded and/or removed with physical and chemical processes, and sometimes the degradation products are more toxic [13]. Currently, one of the possible alternatives for treatment of textile effluents is the use of bacteria or their enzymes, which can oxidize a wide spectrum of synthetic dyes [13]. The demand for removal of synthetic dyes from the textile industrial waste using fugal/bacterial laccase is being increased tremendously. Laccase has been reported as an inducible enzyme during degradation of azo dyes by various bacteria [14,15]. Pseudomonas desmolyticum NCIM 2112 has been reported to decolorize and degrade reactive and benzidine based azo dyes at the static anoxic condition [16,17]. Even though the induction of dye degrading enzymes has been reported with proposed metabolic pathways, the individual role of enzymes is not discussed. There are several reports on effect of physicochemical parameters, cell aging, dye concentration, immobilization of whole cells and consortium on bacterial dye decolorization [15,18,19]. Studies on the action of purified bacterial laccase are scarce and its role in color removal remains poorly understood. To this end, we have purified and characterized the laccase from P. desmolyticum NCIM 2112. The degradation of three textile dyes by purified laccase has been studied.

66

S. Kalme et al. / Enzyme and Microbial Technology 44 (2009) 65–71

2. Materials and methods

2.6. Characterization of purified laccase

2.1. Chemicals

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was carried out following the protocol of Laemmli [25] with 4% (w/v) stacking and 10% (w/v) resolving gel, run on a slab gel unit (Genetech Laboratories, India). Protein bands were stained with Coomassie brilliant blue R-250 at 0.1% (w/v) in methanol/acetic acid/water (v/v/v) (4:1:5) for 1 h at room temperature followed by destaining using same solution. The molecular mass of the purified laccase was determined by calculating the relative mobility of standard protein markers (Genie, India) run alongside (205,000 Da myosin rabbit muscle; 0.5 mg ml−1 , 97,400 Da phosphorylase b; 0.5 mg ml−1 , 66,000 Da bovine serum albumin; 0.5 mg ml−1 , 43,000 Da ovalbumin; 0.75 mg ml−1 , 29,000 Da carbonic anhydrase; 0.5 mg ml−1 , 20,100 Da soyabean trypsin inhibitor; 2.0 mg ml−1 , 14,300 Da lysozyme; 0.75 mg ml−1 ). Zymogram analysis for laccase activity was performed on native-PAGE using 1 mM L-Dopa in 0.1 M acetate buffer (pH 4.9), after washing the gels for 1 h with the same buffer. Substrate specificity of the purified laccase was determined spectrophotometrically at 30 ◦ C in a reaction mixture of 2 ml containing either substrate (5 mM hydroquinone, 5 mM DAB, 2 mM o-tolidine, 0.66 mM ABTS, 5 mM L-dopa) in 0.1 M acetate buffer (pH 4.9) and 100 ␮l enzyme. The rate of oxidation of various substrates catalyzed by purified laccase was determined by measuring the absorbance change with the molar extinction coefficient (ε) obtained from literature [26,27]. The optimum pH for the purified laccase was examined in the pH range 2.0–7.0 (0.1 M of KCl–HCl buffer; pH 2.0, glycine–HCl buffer; pH 3.0, sodium-acetate buffer; pH 4.0–5.0 and sodium-phosphate buffer; pH 5.0–7.0) with 0.66 mM ABTS, and 5 mM 3-3 -diaminobenzidine tetrahydrochloride (DAB) and hydroquinone as a substrate separately. The optimum temperature and the effect of putative laccase inhibitors and metal ions on laccase activity were determined using 5 mM hydroquinone as substrate in 0.1 M sodium-acetate buffer (pH 4.0). For temperature study (30–80 ◦ C), the reaction mixture was incubated at each temperature for 10 min before enzyme addition. The individual inhibitors tested were 1 mM Ethylene diamine tetraacetic acid (EDTA), Dithiothreitol (DTT), and metal ions used at 1 mM concentration were MgCl2 , CaCl2 , HgCl2 , and CuSO4 . The purified laccase was incubated with metal ions and inhibitors for 10 min and the enzyme activity was assayed in triplicate as described above. Control samples were maintained without the metal ions and laccase inhibitors.

DEAE (diethyl aminoethyl) cellulose was purchased from Sisco Research Laboratories, India. Biogel P100 was purchased from Biorad, USA. 2,2 -azinobis (3ethylbenzthiazoline-6-sulfonate (ABTS) was purchased from Sigma–Aldrich, USA. The textile dyes, Direct Blue-6 (DB6; C. I. Direct Blue 6), Green HE4B (GHE4B; C. I. Reactive Green 19A) and Red HE7B (RHE7B; C. I. Reactive Red 141) were the generous gift from local textile industry, Solapur, India. All chemicals used were of the highest purity available and of the analytical grade. 2.2. Microorganism and culture condition P. desmolyticum NCIM 2112 was obtained from National Center for Industrial Microorganisms, NCL, India. As previous studies from our lab have reported induction of laccase activity in P. desmolyticum NCIM 2112 during degradation of reactive and benzidine based dyes [16,17], we selected this strain for the production of laccase. Pure culture was maintained on nutrient agar slants at 4 ◦ C by transferring culture once in a month. Composition of nutrient medium used for decolorization studies was (g l−1 ): NaCl 5, peptone 5, beef extract 3. 2.3. Laccase production Time course of laccase production by P. desmolyticum was studied in 100 ml nutrient broth at 30 ◦ C at static condition. Laccase activity (as mentioned in following section) was measured in crude cell extract of P. desmolyticum cells [20] grown at different time intervals. Dry weight of cells was measured as reported previously [20]. For higher laccase production, 10% inoculum of 12 h (A660 nm 0.7) grown P. desmolyticum was inoculated in 3 l nutrient medium and incubated ∼12 h at 30 ◦ C. Cells were collected by centrifugation at 8000 × g for 15 min and suspended (150 mg ml−1 ) in 50 mM sodium phosphate buffer (pH 7.0; buffer A) containing 5 mg ml−1 lysozyme. Cells were further incubated at 37 ◦ C for 45 min in water bath and then disrupted by sonication as mentioned previously [20]. This cell free extract was solubilized in cholic acid (0.33 mg mg protein−1 ) on magnetic stirrer at 4 ◦ C for 30 min. The cell lysate obtained was centrifuged twice at 15,000 × g for 30 min at 4 ◦ C and the clear supernatant used immediately or stored at −20 ◦ C until its use to purify laccase. 2.4. Purification of laccase All purification steps were carried out using Biorad protein purification system (EP 1-Econo pump) at 4 ◦ C. The supernatant containing laccase activity 0.04 U (mg protein)−1 min−1 was heated at 60 ◦ C for 10 min and centrifuged at 8000 × g for 20 min. The clear supernatant obtained after centrifugation was loaded on a DEAE cellulose fast flow column (15 mm × 120 mm), equilibrated with buffer A. The column was washed with the same buffer by two times of the column volume and the enzyme was eluted with a linear gradient of 0–1.0 M NaCl. Fractions containing laccase activity were pooled and dialyzed against 1 mM sodium phosphate buffer (pH 6.0). The dialyzed sample was concentrated (1–2 ml) by ultrafiltration using YM10 cut-off membrane (Amicon, Boston, MA) and loaded on Biogel P100 column (10 mm × 500 mm) equilibrated with 50 mM sodium phosphate buffer (pH 6.0, buffer B). The protein elution was carried with the same buffer at 6 ml h−1 flow rate. Fractions containing laccase activity were pooled and stored at −20 ◦ C until use.

2.7. Decolorization and degradation of dyes by purified laccase One benzedine based dye, DB6 (MW 932.76; max 540 nm) and two reactive azo dyes, GHE4B (MW 871.07; max 630 nm) and RHE7B (MW 1070; max 552 nm) were used in this study. These dyes are previously reported to be decolorized by P. desmolyticum NCIM 2112 [16,28,17]. The decolorization reaction was carried out at 30 ◦ C for 24 h in 2 ml reactions mixture containing 100 mg l−1 dye prepared in 50 mM acetate buffer (pH 4.8) and 0.5 U ml−1 purified laccase. Control containing heat-denatured enzyme was used to measure decolorization of dye at different time interval. The decolorization was monitored by scanning the UV–vis spectrum between 200–800 nm using Hitachi (U-2800) double beam spectrophotometer. The above decolorized samples were centrifuged at 1000 × g for 1 min and laccase degraded metabolites of DB6, GHE4B and RHE7B were extracted by adding twice equal volume of ethyl acetate. After drying over anhydrous Na2 SO4 , the biodegraded metabolites were characterized by Fourier Transform Infrared Spectroscopy (PerkinElmer, Spectrum one) and compared with control dye. The FTIR analysis was done in the mid IR region of 400–4000 cm−1 with 16 scan speed. The samples were mixed with spectroscopically pure KBr in the ratio of 5:95, pellets were fixed in sample holder, and the analyses were carried out.

2.5. Protein determination and enzyme activity The protein concentration of each fraction was monitored by absorbance at 280 nm or Lowry methods with bovine serum albumin as a standard [21]. Laccase activity was determined at 30 ◦ C by measuring increase in optical density at 420 nm in a reaction mixture of 2 ml containing 0.66 mM ABTS in 0.1 M acetate buffer (pH 4.9) and 100 ␮l enzyme [22]. One unit of enzyme activity was defined as a change in absorbance unit min−1 mg protein−1 . The monophenolase activity of purified protein was determined using p-cresol and 4-hydroxyanisole (4HA) as substrate. Cresolase activity toward p-cresol was measured spectrophotometrically by the appearance of 4-methyl-o-benzoquinone at 400 nm (ε = 1350 M−1 cm−1 ), as has been described by Mayer et al. [23]. The reaction mixture of 2 ml contained 100 ␮l enzyme and 1 mM p-cresol in 100 mM Na-PO4 buffer (pH 7.0) at 30 ◦ C. One unit of enzyme was defined as the amount of enzyme that produced 1 ␮mol of tert-butylquinone min−1 . 4HA was used as substrate to determine the monophenolase activity of purified protein [24]. The 100 ␮l enzyme was incubated with 50 mM sodium acetate, pH 5.5, 2% (v/v) DMF and 50 mM 3methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) at 25 ◦ C for 5 min. The reaction was started by addition of 100 mM 4HA and monitored spectrophotometrically at 500 nm. One unit of enzyme activity (U) is defined as the amount of enzyme producing 1 ␮mol of MBTH-quinone adducts per minute during the linear phase of the reaction.

3. Results 3.1. Purification of laccase P. desmolyticum achieved maximum intracellular laccase activity of 0.012 ± 0.0003 U mg−1 protein in the crude cell free extract of 24 h growth (Fig. 1). Laccase production was more between 18 and 24 h of incubation period. Although dry cell weight increased after 24–36 h incubation period, significant loss in the laccase activity was observed. The purification of laccase from P. desmolyticum consisted of three steps including heating, anion exchange and molecular sieve chromatography. Heating of cell free extract at 60 ◦ C for 10 min proved to be effective for removing large amounts of proteins as laccase was stable for this period and there was no significant loss of the enzyme activity. The specific activity of laccase was increased from 0.04 to 0.081 U mg−1 proteins on heating of the cell free extract. Further it was increased to 0.424 U mg−1

S. Kalme et al. / Enzyme and Microbial Technology 44 (2009) 65–71

67

Table 2 Substrate oxidizing activity of purified laccase. Substrate

Absorbance (nm)

Relative activity (%)

Hydroquinone DAB o-tolidine ABTS L-dopa

250 410 366 420 475

100 34 20 13 7

Relative activity was calculated from the specific activity of each substrate.

laccase (Fig. 2, lane D) and protein staining showed molecular mass similar to that of reported by SDS–PAGE (Fig. 2, lane E). 3.2. Substrate specificity of laccase

Fig. 1. Time course evolution of P. desmolyticum 2112 growth in form of dry weight and laccase production in static condition at 37 ◦ C in 100 ml nutrient broth.

Several typical laccase substrates were oxidized by a purified enzyme as indicated by an intense increase of optical density measured at appropriate wavelength. The conventional substrates of laccase such as hydroquinone, DAB, o-tolidine, ABTS and Ldopa were oxidized and their relative activities are as given in Table 2. Purified laccase oxidizes hydroquinone maximally among the different substrates tested. In relation to hydroquinone the substrate specificity of laccase was in the following order: DAB > otolidine > ABTS > L-dopa. The fractions containing purified laccase did not show monophenolase activity. 3.3. pH and temperature dependency Three substrates were used to determine the effect of pH on laccase activity as laccase enzymes tend to react differently to pH with different substrates [29]. The pH optima obtained for P. desmolyticum laccase was in the acidic region and representative of typical laccases. The optimal pH for DAB oxidation was 4.5, and that of ABTS and hydroquinone was 4. The enzyme exhibited a comparatively broader pH (3.0–7.0) profile with hydroquinone than the other substrates tested (Fig. 3). The effect of temperature on laccase activity showed that it is active in the temperature range from 30 to 65 ◦ C with the maximum activity at 60 ◦ C (Fig. 4). The temperature range for active enzyme is remarkably wide.

Fig. 2. SDS–PAGE (lane A–C) and native PAGE (lane D–F) analysis of purified laccase. Lane A, molecular size markers; lane B, supernatant of cell free extract; lane C, laccase purified by Biogel P100; lane D, L-dopa stained zymograph of laccase; lane E, protein staining of laccase; lane F, molecular size markers. For native PAGE analysis (lane D and E) DEAE eluted laccase was used.

proteins after passing through DEAE cellulose column. The anion exchange resin (DEAE) served to further separate the laccase activity from other proteins present in the fraction. The purification fold and yield of laccase obtained through Biogel P100 were 45.75 and 19%, respectively. The purification steps and relevant details such as laccase activity, specific activity, percent yield and fold purification are outlined in Table 1. The enzyme obtained after molecular sieve chromatography showed single protein band on SDS–PAGE with molecular mass of 43 kDa (Fig. 2, lane C). Staining of native gel with L-dopa showed dark brown color band indicating presence of

3.4. Effect of inhibitors and metal ions EDTA inhibited laccase activity by 97% whereas total inhibition of the activity was observed in the presence of DTT. The effect of metal ion showed that the laccase activity was inhibited by CaCl2 (12%), HgCl2 (20%), MgCl2 (51%) and complete inhibition by CuSO4 (data not shown). 3.5. Decolorization and degradation of textile dyes by purified laccase During in vitro decolorization study, DB6 and RHE7B were decolorized by 100% within 16 h whereas GHE4B took 12 h to decolorize completely. The heat-denatured enzyme (control) did not support color removal, but in the presence of laccase, significant modification occurred in the UV–vis absorbance spectrum of all dyes. Fig. 5

Table 1 Summary of purification of laccase from P. desmolyticum. Purification step

Volume (ml)

Total protein (mg)

Total activity (U)

Specific activity (U mg−1 )

Purification (fold)

Yield (%)

Supernatant of cell free extract Heated supernatant DEAE cellulose Biogel P100

8 7.5 10.4 2

143 71 5 0.60

5.85 5.80 2.12 1.10

0.040 0.081 0.424 1.83

1 2.02 10.6 45.75

100 99 36 19

68

S. Kalme et al. / Enzyme and Microbial Technology 44 (2009) 65–71

Fig. 3. pH optima for laccase with various substrates as well as the pH range of oxidation for each of the substrates. Error bars are ±S.E.M. (standard error of mean) of three independent experiments.

Fig. 4. Temperature optima for laccase in the presence of hydroquinone. Error bars are ±S.E.M. of three independent experiments.

depicts 51% (6 h), 56% (9 h) and 65% (9 h) decolorization of GHE4B, DB6 and RHE7B respectively. In fact, almost complete removal of the major peak at their max was accompanied by reduction of absorbance in the visible region (400–800 nm) and there was not appearance of any new peak in visible region except increased absorbance towards the UV region (200–400 nm). In the UV spectra, three new peaks emerged at 280, 290 and 320 nm. Peak at 320 nm and a typical absorption at 245–250 nm were reported in decolorized samples of all three dyes. However, peak at 280 nm was observed in GHE4B decolorized sample and peak at 290 nm was observed in DB6 decolorized sample.

Fig. 5. UV–vis absorbance spectrum of undecolorized dyes (A) GHE4B, (B) RHE7B and (C) DB6 and dyes decolorized in the presence of purified laccase. Control reactions were without or heat-denatured laccase.

Table 3 FTIR analysis of control dye and dye metabolites formed after laccase treatment. Dye

Control peaks (cm−1 )

New peak (cm−1 )

Bonds susceptible to laccase (cm−1 )

DB6

640 (S O s), 977 (C O s), C N s: (1044 asy, 1186 sy, 1340), N H s: (1492, 3421), 1615 ( N N s), 2924 (C H s) 550-713 (C S s), 1017, 1041 (S O s; asy), 1135 (C N s; sy), 1340, 1489 (N H s), 1574 ( N N s) 617–976 (H adjacent to naphthalene), 1411 (O H def), 1041 and 1206 (S O s; sy), 1541 (N H def + C N s), 2924, 3421 (N H s at pyrole ring like)

1017 (C OH s)

640, 977, 1186, 1340, 1492 and 1615

1083, 1647 3401

713–844 (C S s), 1135, 1489 and 1574

1019, 1600

617–976, 1041, 1206, 1411, 1470 and 1541

GHE4B RHE7B

s, Stretching; sy, symmetric; asy, asymmetric; def, deformation.

S. Kalme et al. / Enzyme and Microbial Technology 44 (2009) 65–71

69

The FTIR spectral comparison between the control dye sample and the samples extracted after decolorization by purified laccase have confirmed degradation of these dyes (Figs. 6 and 7). In control dye spectrum, peak at 1340 cm−1 represents C N stretching of aromatic amine group in DB6 (Fig. 6) and GHE4B (Fig. 7A and B). The free NH stretching in DB6 was reported at 1492 and 3421 cm−1 represents N H stretching of pyrrole ring like structure in RHE7B (Fig. 7C and D). The presence of sulfonic acid was confirmed by asymmetric S O stretching at 1041 and 640 cm−1 in RHE7B/GHE4B and DB6, respectively. Appearance of some new peaks and absence of important peaks required for structural integrity of the dyes have been observed in the FTIR analysis of the metabolites produced after decolorization (Table 3). 4. Discussion

Fig. 6. FTIR analysis of (A) DB6 dye and (B) DB6 metabolites produced after decolorization of DB6 by purified laccase.

Laccase is a phenol oxidase (PO) that can oxidize o- and p-diphenols, and does not have cresolase (o-hydroxylation of monophenols) activity. On the contrary, phenoloxidases of the tyrosinase type have both cresolase and catecholase activity [30]. The absence of monophenolase activity in purified fractions containing laccase has excluded the presence of other types of phenoloxidades. The molecular mass of the purified P. desmolyticum laccase (43 kDa) is different than some recently characterized laccase from P. putida F6 strain [13] and Marinomonas mediterranea [31], whereas, one of the laccase isozyme in Azospirillum lipoferum [12] has nearly same molecular weight (48.9 kDa). In P. desmolyticum, the optimum pH (4.0) for laccase activity was in acidic region when compared to other bacterial laccases which ranges at 5–8. P. desmolyticum laccase showed bell shaped behavior similar to the Trametes sp. strain AH28-2 [32] and Pleurotus sajor-caju [33] laccases involved in dye decolorization. Though the enzyme is a Cu containing protein, inhibition by Cu was also

Fig. 7. FTIR analysis of (A) GHE4B dye, (B) GHE4B metabolites, (C) RHE7B dye and (D) RHE7B metabolites produced after decolorization of RHE7B by purified laccase.

70

S. Kalme et al. / Enzyme and Microbial Technology 44 (2009) 65–71

reported in the laccase from Lentinus edodes [34] and P. ostreatus [35]. The time course of induction of oxidative enzymes during degradation of DB6 [16] and RHE7B in P. desmolyticum is previously reported [17]. In both the studies laccase was significantly induced with LiP activity. Since, purified laccase under long incubation conditions decolorizes all three dyes, but heat-denatured enzyme does not, it indicates that laccase is involved in color removal and corroborates recent observations [36,37]. In GHE4B and RHE7B decolorized samples, the appearance of new peaks at 280 and 320 nm indicated degradation of dyes (azo conjugated structure) into low molecular weight aromatic compounds. The formation of a new peak at 290 nm suggests that there were changes in the aromatic group in DB6 decolorized sample. In all three dyes some degraded products have hydrogenated azo bond structures [16,17,28] and those structures showed a typical absorption peak at 245–250 nm indicating only a partial azo bond disruption [38]. Absence of C N symmetric stretching in DB6 and GHE4B metabolites and N Hdef + C NS in RHE7B metabolites indicates the asymmetric cleavage of these dyes. Further breakdown of azo bond possessing intermediates by oxidation–reduction [16] can be supported by susceptible peaks at 1615 cm−1 in DB6 and 1574 cm−1 in GHE4B. The degradation product of GHE4B was identified as 4-amino, 6-hydroxynaphthalene, 2-sulfonic acid by high pressure liquid chromatography [28]. The formation of this product could be possible by asymmetric cleavage and FTIR results support this phenomenon, as metabolites showed NH deformation + CN stretching in primary amines at 1647 cm−1 and N H stretching of primary amines at 3401 cm−1 . Decreased intensity of peaks from fingerprint region and new peak at 1083 cm−1 indicates formation of mono or di-substituted compounds, as GHE4B metabolites. Recently we have reported desulfonation of RHE7B metabolites before and after ring cleavage by P. desmolyticum whole cells [17]. In this study, FTIR data obtained after RHE7B degradation by purified laccase, where peak for S O and O H group, conferring the character of a laccase substrate were found to be susceptible (Table 3). Chivukula and Renganathan [39] have observed oxidation of phenolic dyes having electron donating methyl or methoxy substituents by laccase. The sulfo and hydroxyl groups were in para-positions of the respective phenyl rings. Martins et al. [40] reported a positional effect as the dyes containing sulfonic group in the para position to the azo link were decolorized to the greater extent than its meta substituted analogue. Soares et al. [41] also found that dye containing sulfonic acid group was readily transformed by the laccase. Azo dyes sulfonated in the meta position or carrying a hydroxyl group in the ortho-position to the azo bond were not taken into consideration. Although a large number of structurally related dyes can be successfully oxidized by laccases, decolorization take place at different rates and to different extents and many dyes are not degraded at all [42]. In this study, GHE4B was decolorized faster as compare to RHE7B and DB6 by purified laccase. FTIR analysis suggests that laccase has preferred the asymmetric cleavage of azo bond in all the dyes, but the degree of specificity was more for the dye containing sulfonic group and amino group at meta position to the azo bond present in it (GHE4B). Whereas dyes having sulfonic group and hydroxyl group at meta position to the azo bond showed lower extent decolorization with respect to time (RHE7B and DB6). Substituents of the dyes like the hydroxyl groups or the amino groups are more likely to be degraded than dyes with a methyl, methoxy, sulfo or nitro group [43] and the effect of sulfonates present in reactive or direct dyes is varied for levels of color removal [44]. However, the direct dye decolorization is usually not correlated to the number of sulfonate groups in the dye structure [45]. In this study although the number of sulfonate groups (8) in GHE4B and RHE7B (both are reactive

dyes) are same, the removal of dye was reported higher in GHE4B than RHE7B, so it is hypothesized that, the position of sulfonic and amino/hydroxy group in a dye would render its availability to the laccase. From this study it is clear that the substitution pattern on these dyes contributed major role towards their biodegradability. Further, there is correlation between redox potential of the dyes and decolorization rates [46]. However, complementary studies are required to study a relationship between the redox potential of the azo dyes and the decolorization efficiency of P. desmolyticum laccase, laccase/mediator, and bacterial cells, besides further information on the mechanisms involved in the degradation of these dyes. This study on application of P. desmolyticum laccase in degradation of dyes and focus on the structural requirement of a dye to be biodegradable is important in order to optimize potential bioremediation systems for industrial textile process water treatment. Acknowledgement Authors acknowledge FTIR facility by Common Facility Center, Shivaji University, Kolhapur. References [1] Ghodake GS, Kalme SD, Jadhav JP, Govindwar SP. Purification and partial characterization of lignin peroxidase from Acinetobacter calcoaceticus NCIM 2890 and its application in decolorization of textile dyes. Appl Biochem Biotechnol 2008, doi:10.1007/s12010-008-8258-4. [2] Couto RS, Herrera JT. Industrial biotechnological applications of laccases: a review. Biotechnol Adv 2006;24:500–13. [3] Mayer AM, Staples RC. Laccase: new functions for an old enzyme. Phytochemistry 2002;60:551–65. [4] Claus H. Laccase and their occurrence in prokaryotes. Arch Microbiol 2003;179:145–50. [5] Thurston CF. The structure and function of fungal laccases. Microbiology (UK) 1994;140:19–26. [6] Baldrian P. Purification and characterization of laccase from the white-rot fungus Daedalea quercina and decolorization of synthetic dyes by the enzyme. Appl Microbiol Biotechnol 2004;63:560–3. [7] Ryan S, Schnitzhofer W, Tzanov T, Cavaco-Paulo A, Gübitz GM. An acid-stable laccase from Sclerotium rolfsii with potential for wool dye decolourization. Enzyme Microb Technol 2003;33:766–74. [8] Murugesan K, Nam I-H, Kim Y-M, Chang Y-S. Decolorization of reactive dyes by a thermostable laccase produced by Ganoderma lucidum in solid state culture. Enzyme Microb Technol 2007;40:1662–72. [9] Zouari-Mechichi H, Mechichi T, Dhouib A, Sayadi S, Martínez AT, Martínez MJ. Laccase purification and characterization from Trametes trogii isolated in Tunisia: decolorization of textile dyes by the purified enzyme. Enzyme Microb Technol 2006;39:141–8. [10] Litthauer D, Jansen van Vuuren M, Van Tonder A, Wolfaardt FW. Purification and kinetics of a thermostable laccase from Pycnoporus sanguineus (SCC 108). Enzyme Microb Technol 2007;40:563–8. [11] Diamantidis G, Effosse A, Rene P, Bally P. Purification and characterization of the first bacterial laccase in the rhizospheric bacterium Azospirillum lipoferum. Soil Biol Biochem 2000;32:919–27. [12] McMahon AM, Doyle EM, Brooks S, O’Connor KE. Biochemical characterisation of the coexisting tyrosinase and laccase in the soil bacterium Pseudomonas putida F6. Enzyme Microb Technol 2007;40:1435–41. [13] Robinson T, McMullan G, Marchant R, Nigam P. Remediation of dyes in textile effluent: a critical review on current treatment technologies with a proposed alternative. Bioresour Technol 2001;77:247–55. [14] Parshetti G, Kalme S, Saratale G, Govindwar S. Biodegradation of malachite green by Kocuria rosea MTCC 1532. Acta Chim Slov 2006;53:492–8. [15] Kalyani DC, Patil PS, Jadhav JP, Govindwar SP. Biodegradation of reactive textile dye Red BLI by an isolated bacterium Pseudomonas sp. SUK1. Bioresour Technol 2008;99:4635–41. [16] Kalme SD, Parshetti GK, Jadhav SU, Govindwar SP. Biodegradation of benzidine based dye direct blue-6 by Pseudomonas desmolyticum NCIM 2112. Bioresour Technol 2007;98:1405–10. [17] Kalme S, Ghodake G, Govindwar S. Red HE7B degradation using desulfonation by Pseudomonas desmolyticum NCIM 2112. Int Biodet Biodeg 2007;60:327–33. [18] McMullan G, Meehan C, Conneely A, Kirby N, Robinson T, Nigam P, et al. Microbial decolorization and degradation of textile dyes. Appl Microbiol Biotechnol 2001;56:81–7. [19] Fang H, Wenrong H, Zhong LY. Biodegradation mechanisms and kinetics of azo dye 4BS by a microbial consortium. Chemosphere 2004;57:293–301. [20] Kalme SD, Parshetti GK, Gomare SS, Govindwar SP. Diesel and kerosene degradation by diesel and kerosene degradation by Pseudomonas desmolyticum

S. Kalme et al. / Enzyme and Microbial Technology 44 (2009) 65–71

[21] [22]

[23] [24]

[25] [26]

[27]

[28] [29]

[30]

[31]

[32]

NCIM 2112 and Nocardia hydrocarbonoxydans NCIM 2386. Curr Microbiol 2008;56:581–6. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265–75. Hatvani N, Mecs I. Production of laccase and manganese peroxidase by Lentinus edodes on malt containing by product of the brewing process. Process Biochem 2001;37:491–6. Mayer AM, Harel E, Ben-Shaul R. Assays of catechol oxidase. A critical comparison of methods. Phytochemistry 1966;5:783–6. Dicko MH, Gruppen H, Barro C, Traore AS, Berkel WJH, Voragen AGJ. Impact of phenolic compounds and related enzymes in Sorghum varieties for resistance and susceptibility to biotic and abiotic stresses. J Chem Ecol 2005;31:2671–88. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680–5. Eggert C, Temp U, Eriksson K-EL. The lignolytic system of the white rot fungus Pycnoporus cinnabarinus: purification and characterization of the laccase. Appl Environ Microbiol 1996;62:1151–8. Shin K-S, Lee Y-J. Purification and characterization of a new member of the laccase family from the white-rot basidiomycete Coriolus hirsutus. Arch Biochem Biophys 2000;384:109–15. Kalme S. Role of microbes in biotransformation of chemicals. Ph.D. Thesis. Shivaji University, Kolhapur, India; 2006. p. 111–2. Jordaan J, Pletschke BI, Leukes WD. Purification and partial characterization of a thermostable laccase from an unidentified basidiomycete. Enzyme Microb Technol 2004;34:635–41. Pérez-Gilabert M, Carmona FG. Characterization of catecholase and cresolase activities of eggplant polyphenol oxidase. J Agric Food Chem 2000;48:695– 700. ´ Jimenez-Juarez N, Roman-Miranda R, Baeza A, Sanchez-Amat A, VazquezDuhalt R, Valderrama B. Alkali and halide-resistant catalysis by the multipotent oxidase from Marinomonas mediterranea. J Biotechnol 2005;117:73–82. Xiao YZ, Tu XM, Wang J, Zhang M, Cheng Q, Zeng WY, et al. Purification and characterization of laccase produced by a white rot fungus Pleurotus sajorcaju under submerged culture condition and its potential in decolorization of azo dyes with aromatic compounds of a laccase. Appl Microbiol Biotechnol 2003;60:700–7.

71

[33] Murugesan K, Arulmani M, Nam I-H, Kim Y-M, Chang Y-S, Kalaichelvan PT. Purification and characterization of laccase produced by a white rot fungus Pleurotus sajor-caju under submerged culture condition and its potential in decolorization of azo dyes. Appl Microbiol Biotechnol 2006;72:939–46. [34] Kofujita H, Ohte T, Asada Y, Kuuahara M. Purification and characterization of laccase from Lentinus edodes. Mokuz ai Gakkalshi 1991;37:562–9. [35] Okamoto K, Yanagi SO, Sakai T. Purification and characterization of extracellular laccase from Pleurotus ostreatus. Mycoscience 2000;41:7–13. [36] Schliephake K, Mainwaring DE, Lonergan GT, Jones IK, Baker WL. Transformation and degradation of the disazo dye chicago sky blue by a purified laccase from Pycnoporus cinnabarinus. Enzyme Microb Technol 2000;27:100–7. [37] Nagai M, Sato T, Watanabe H, Saito K, Kawata M, Enei H. Purification and characterization of an extracellular laccase from the edible mushroom Lentinula edodes, and decolorization of chemically different dyes. Appl Microbiol Biotechnol 2002;60:327–35. [38] Qing C. Chemical industry. In: Chemistry of dye intermediates. China: Press Beijing; 1989. p. 133. [39] Chivukula M, Renganathan V. Phenolic azo dye oxidation by laccase from Pyricularia oryzae. Appl Environ Microbiol 1995;61:4374–7. [40] Martins MAM, Ferreira LC, Santos LM, Queiroz MJ, Lima N. Biodegradation of bioaccessible textile azo dyes by Phenerochaete chrysosporium. J Biotechnol 2001;89:91–8. [41] Soares GMB, Amorim MTP, Hrdina R, Costa-Ferreira M. Studies on biotransformation of novel disazo dyes by laccase. Process Biochem 2002;37:581–7. [42] Kandelbauer A, Maute O, Kessler RW, Erlacher A, Gubitz GM. Study of dye decolorization in an immobilized laccase enzyme-reactor using online spectroscopy. Biotechnol Bioeng 2004;87:552–63. [43] Nigam P, Banat IM, Singh D, Marchant R. Microbial process for the decolorization of textile effluent containing azo, diazo and reactive dyes. Proc Biochem 1996;31:435–42. [44] Pandey A, Singh P, Iyengar L. Bacterial decolorization and degradation of azo dyes. Int Biodet Biodeg 2007;59:73–84. [45] Hitz HR, Huber W, Reed RH. The absorption of dyes on activated sludge. J Soc Dyers Color 1978;94:71–6. [46] Zille A, Ramalho P, Tzanov T, Millward R, Aires V, Cardoso MH, et al. Predicting dye biodegradation from redox potentials. Biotechnol Prog 2004;20:1588–92.