Biotechnology and Bioprocess Engineering 2009, 14: 369-376 DOI/10.1007/s12257-008-0300-4
Purification and Characterization of Veratryl Alcohol Oxidase from Comamonas sp. UVS and Its Role in Decolorization of Textile Dyes Umesh U. Jadhav1*, Vishal V. Dawkar1, Dhawal P. Tamboli2, and Sanjay P. Govindwar1 1
Department of Biochemistry, Shivaji University, Kolhapur 416-004, India Department of Biotechnology, Shivaji University, Kolhapur 416-004, India
2
=
^Äëíê~Åí= In the present work, we have purified veratryl alcohol oxidase (VAO) enzyme from `çã~ãçå~ë sp. UVS to evaluate its potential to decolorize textile dyes. VAO was purified (13.9 fold) by an ion exchange followed by the size exclusion chromatography. Molecular weight of the VAO was estimated to be about 66 kDa by SDS-PAGE. The optimum pH and temperature of oxidase were 30°C and 65°C, respectively. VAO showed maximum activity with n-propanol among the various substrates (å-propanol, veratryl alcohol, L-dopa, tryptophan, ÉíÅ.). Under standard assay conditions, Km value of the enzyme was 2.5 mM towards veratrole. The enzyme activity was completely inhibited by 0.5 mM sodium azide. L-cysteine, dithiothreitol, and the metal chelator, EDTA had a slight inhibitory effect. The purified enzyme was able to decolorize textile dyes, Red HE7B (57.5%) and Direct Blue GLL (51.09%) within 15 h at 40 µg/mL concentration. GC-MS analysis of the metabolites suggested oxidative cleavage and desulphonation of these dyes. © KSBB hÉóïçêÇëW=sÉê~íêóä=~äÅçÜçä=çñáÇ~ëÉI=Comamonas=ëéK=rspI=ÇóÉ=ÇÉÅçäçêáò~íáçåI=ãÉí~ä=áçåëI=d`Jjp
INTRODUCTION Dyes are released into the environment as the industrial effluents from two major sources, the textile and the dyestuff industries [1]. A necessary criterion for the use of these dyes is that they must be highly stable in light and during washing [2]. They must also be resistant to microbial attack. Therefore, they are not readily degradable and are typically not removed from water by conventional wastewater treatment systems [3]. In recent years a great deal of research has been directed towards developing processes in which enzymes are employed to remove/transform phenolic compounds and dyes from polluted wastewater [4,5]. Many researchers have shown that the effective degradation and precipitation of industrially important azo dyes was catalyzed by HRP (Horseradish peroxidase), plant peroxidases, manganese peroxidases (MnP), laccase, etc. [6-12]. Dyes and phenols are oxidized by peroxidases to generate phenoxy radicals, which couple to form oligomeric and polymeric products [13,14]. These polymeric aggregates have G`çêêÉëéçåÇáåÖ=~ìíÜçê= Tel: +91-231-2609152 Fax: +91-231-2691533 e-mail:
[email protected]
limited solubility and tend to precipitate quite readily. The increase in the molecular weight and precipitation of the product is thought to be accompanied by a detoxification effect. This will allow a possible alternative for the treatment of industrial wastewater to reach sub ppm levels for which conventional methods are ineffective. Bioremediation is becoming important because of cost effectiveness, environmental friendliness, and production of less sludge as compare to the physicochemical decomposition processes [15,16]. Peroxidase catalyzes a variety of oxidation reactions. Oxidases are capable of oxidizing a variety of xenobiotic compounds including polycyclic aromatic hydrocarbons, polychlorinated phenols, nitroaromatics, and azo dyes [17]. A majority of the work on degradation of dyestuffs by whole cultures, crude enzyme preparations, and purified ligninolytic enzymes have been carried out for the decolorization of the different dyes using microorganisms. In present study, an attempt has been made to purify veratryl alcohol oxidase (VAO) from Comamonas sp. UVS and its characteristics like temperature, pH, and substrate optima. In addition, the role of oxidase in decolorization of structurally different groups such as azo, thiazin, heterocyclic, and polymeric dyes were studied.
PTM=
MATERIALS AND METHODS
dase bands.
`ìäíáî~íáçå=çÑ=`çã~ãçå~ë=ëéK=rsp=~åÇ=pãéäÉ= = mêÉé~ê~íáçå=
aÉíÉêãáå~íáçå=çÑ=léíáãìã=ée=~åÇ=qÉãéÉê~íìêÉ=Ñçê= = s^l=båòóãÉ
Comamonas sp. UVS was grown in the yeast extract medium up to its exponential phase. Cells were collected by centrifugation at 8,000 g for 20 min and suspended in 0.05 M potassium phosphate buffer (pH 7.4) (1 g cells/mL). Cells were then disrupted by sonication. This cell free extract was centrifuged at 10,000 g for 20 min at 4°C. The clear supernatant was collected and used as crude source of VAO enzyme.
VAO enzyme activity, as a function of pH, was determined by using veratryl alcohol as substrate, in buffers ranging from pH 1.0 to 8.0. HCl-KCl buffer (0.05 M) was used for pH 1.0~2.0. Citrate phosphate buffer (0.05 M) was used for pH 3.0~5.0 and sodium phosphate buffer (0.05 M) was used for pH 6.0~8.0. VAO activity as a function of temperature was determined at various temperatures from 20~70°C .
mêçíÉáå=aÉíÉêãáå~íáçå=~åÇ=båòóãÉ=^Åíáîáíó=
pìÄëíê~íÉ=péÉÅáÑáÅáíó=~åÇ=båòóãÉ=háåÉíáÅë
The protein concentration of fractions collected during column chromatography was monitored by the absorbance at 280 nm. VAO activity was determined by using veratryl alcohol as a substrate. The reaction mixture contained 4 mM veratryl alcohol, in 0.05 M citrate phosphate buffer, pH 3, and enzyme (6.8 mg) in a total volume of 2 mL was used for the determination of oxidase activity. Oxidation of the substrate at room temperature was monitored by an absorbance increase at 310 nm due to the formation of veratraldehyde [18]. One unit of an enzyme activity was defined as a change in absorbance unit/min/mg protein.
Substrate specificity was determined by using eight different substrates (n-propanol, veratryl alcohol, ethanol, L-dopa, indole, hydroxyquinone, tryptophan, and xylidine). All substrate solutions were prepared in 0.05 M citrate phosphate buffer, pH 3.0. Michaelis constant (Km) and maximum rates (Vmax) were determined by using veratryl alcohol in the range of concentrations 2~10 mM at pH 3.0 and 65°C . The reaction was followed in a spectrophotometer (Hitachi U2800) and data were plotted according to Lineweaver-Burk.
mìêáÑáÅ~íáçå=píÉéë=
The effects of several metal ions (BaCl2, CaCl2, NaCl, MgCl2, KCl, MnCl2, CdCl2, HgCl2, ZnCl2, and CuCl2) on VAO activity were studied. To determine the effect, reactions containing 4 mM veratryl alcohol and 0.098 mg enzyme in 0.05 M citrate phosphate buffer (pH 3.0), were run at 65°C in the presence and absence of above mentioned metal ions. VAO activities were measured at 0.5 mM concentration of the inhibitors. The results were reported as relative activity.
píÉéJNK=ab^bJÅÉääìäçëÉ= = Efçå=ÉñÅÜ~åÖÉ=ÅÜêçã~íçÖê~éÜóF
The clear supernatant obtained after centrifugation was put on to a DEAE-cellulose column (2 × 20 cm) equilibrated with sodium phosphate buffer (pH 7.4, 0.05 M). The column was first washed with NaPO4 buffer of pH 7.0 (76 mL) and then with 0.3 M NaCl (58 mL). The VAO enzyme was eluted with 0.5 M NaCl. Fractions containing oxidase activity were pooled and dialyzed against sodium phosphate buffer (pH 7.0). píÉéJOK=_áçÖÉä=mJNMM= = EpáòÉ=ÉñÅäìëáçå=ÅÜêçã~íçÖê~éÜóF=
The dialyzed sample was concentrated with the help of lyophilizer (Operon, Kyeonggi-DO, Korea) and put on a Biogel P-100 column (1.8 × 50 cm) equilibrated with 0.05 M sodium phosphate buffer (pH 7.0) and eluted with same buffer at a flow rate of 5 mL/h. Fractions containing oxidase activity were pooled and stored at -20°C until use.
bÑÑÉÅí=çÑ=jÉí~ä=fçåë=çå=lñáÇ~ëÉ=^Åíáîáíó=
bÑÑÉÅí=çÑ=fåÜáÄáíçêë=çå=s^l=^Åíáîáíó=
The effects of several inhibitors (Sodium azide, L-cysteine, EDTA, and Dithiothreitol) on VAO activity were studied. To determine the effect, reactions containing 4 mM veratryl alcohol and 0.098 mg of enzyme in 0.05 M citrate phosphate buffer (pH 3.0), were run at 30°C in the presence and absence of above mentioned inhibitors. VAO activities were measured at 0.5 mM concentration of the inhibitors. The results were reported as relative activity. aÉÅçäçêáò~íáçå=çÑ=qÉñíáäÉ=aóÉë=
mçäó~Åêóä~ãáÇÉ=dÉä=bäÉÅíêçéÜçêÉëáë=Em^dbF=^å~äóëáë=
Protein with VAO activity was evaluated with native PAGE (polyacrylamide gel electroporesis). The gels were stained with coomassie brilliant blue R-250 at 0.1% (w/v) in methanol/acetic acid/water (4:1:5, v/v) for 1 h at room temperature followed by destaining using methanol/acetic acid/water (4:1:5, v/v). The native PAGE gel was incubated with 10 mM L-dopa in distilled water for 1 h to develop oxi-
Two textile dyes used in textile industry (Reactive Red HE7B and Direct Blue GLL) were selected and their solutions were prepared in the concentration range of 200 mg/L in distilled water. Each dye was incubated with an oxidase enzyme (2.4 U/mg protein/min) in 0.05 M phosphate buffer, pH 7.0 at 30°C. The disappearance of the color by VAO enzyme was monitored at predetermined λmax of the respective dye solution. The percent decolorization was calculated
Biotechnol. Bioprocess Eng. PTN=
q~ÄäÉ=NK Purification of VAO enzyme from `çã~ãçå~ë=sp. UVS Purification step
Volume (mL)
Total protein (mg)
Total activity (U)
Specific activity (U/mg)
Purification fold
Yield (%)
10
340
21.85
0.064
1
100
Cell lysate (supernatant) DEAE cellulose
2
1.40
0.90
0.642
10.03
4.11
Biogel P-100
1
0.49
0.44
0.890
13.90
2.01
by taking the maximum absorbance of each untreated dye solution as control (100%). The optical density was measured using Hitachi U-2800 spectrophotometer (Tokyo, Japan). Also the decolorization of dyes at various pH (1~8) was also studied. aÉÅçäçêáò~íáçå=~åÇ=aÉÖê~Ç~íáçå=píìÇáÉë=
After decolorization of dyes, the decolorized medium was centrifuged at 5,000 g for 20 min and the supernatant obtained was used to extract metabolites with equal volume of ethyl acetate. The extracts were dried over anhydrous Na2SO4 and evaporated to dryness in rotary evaporator. The crystals obtained were dissolved in small volume of HPLC grade methanol and used for analytical studies. rsJsfp=péÉÅíê~ä=^å~äóëáë=
Decolorization was monitored by UV-Vis spectroscopic analysis (Hitachi U-2800). The culture supernatant obtained after complete decolorization was used for UV-Vis analysis. eáÖÜ=mÉêÑçêã~åÅÉ=iáèìáÇ=`Üêçã~íçÖê~éÜó=Eemi`F=
High performance liquid chromatography (HPLC) analysis was carried out (Waters model no. 2690, Waters corp., Milford, MA) on C18 column (symmetry, 4.6 × 250 mm) by using gradient of methanol and acetonitrile (75:25) with flow rate of 1 mL/min for 10 min. The UV detector was kept at 254 nm for Red HE7B and Direct Blue GLL. d~ë=`Üêçã~íçÖê~éÜóJj~ëë=péÉÅíêçëÅçéó=Ed`JjpF=
GC-MS analysis of metabolites was carried out using a Shimadzu 2010 MS Engine, equipped with integrated gas chromatograph with a HP1 column (60 m long, 0.25 mm id, and nonpolar). Helium was used as a carrier gas at a flow rate of 1 mL/min. The injector temperature was maintained at 280°C with oven conditions as: 80°C kept constant for 2 min-increased upto 200°C with 10°C/min-raised upto 280°C with 20°C/min rate. The compounds were identified on the basis of mass spectra using quadrapole detector and the NIST library.
RESULTS AND DISCUSSION mêÉé~ê~íáçå=çÑ=båòóãÉ=
Comamonas sp. UVS was grown in the yeast extract broth
cáÖK=NK SDS-PAGE of the purified VAO enzyme. A, marker protein; B, purified enzyme; C, Activity staining of purified VAO enzyme with L-Dopa; MW, molecular weight.
(pH 6.5) at 40°C. Then, the cells were collected by centrifugation and suspended in phosphate buffer (pH 7.0, 0.05 M). These cells were sonicated to break the cell wall and to release an enzyme (VAO). Total 10 mL of the centrifuged crude cell free extract having about 340 mg of protein (approximately 21.5 units of VAO activity) was loaded onto the DEAE cellulose (pre-equilibrated with 0.05 M Na-PO4 buffer of pH 7.0). The column was first washed with 0.05 M Na-PO4 buffer of pH 7.0 (76 mL) and then with 0.3 M NaCl (58 mL). VAO was eluted with 0.5 M NaCl. Fractions containing the major peak were pooled and concentrated using lyophilizer, and loaded on the gel filtration column. Then the enzyme was eluted with sodium phosphate buffer (pH 7.0, 0.05 M). An enzyme preparation with a 14 fold increase in specific activity was obtained. The steps for purification of VAO enzyme are summarized in Table 1. The procedure yielded 0.49 mg protein from 10 mL culture filtrate and recovery of total oxidase activity was 2.01%. jçäÉÅìä~ê=tÉáÖÜí=aÉíÉêãáå~íáçå=~åÇ=péÉÅíê~ä= = mêçéÉêíáÉë=çÑ=íÜÉ=mìêáÑáÉÇ=s^l=båòóãÉ
The purified VAO enzyme appeared as a single band on SDS-PAGE and the molecular weight was estimated to be about 66 kDa (Fig. 1). This was further confirmed by activity staining with L-Dopa on native PAGE (Fig. 1). This molecular weight is almost the same as that of aryl alcohol oxidase (AAO) produced by dye-decolorizing fungus, Geotrichum candidum Dec-l [19], but is different from that of other aryl-
PTO=
q~ÄäÉ=OK= Substrate specificity of VAO enzyme Sr. no.
Substrate
1
å-propanol
Specific activity (U/mg protein/min) 1.64
2
Veratrole
1.43
3
Ethanol
0.48
4
L-dopa
0.36
5
Indole
0.34
6
Hydroxyquinone
0.34
7
Tryptophan
0.28
8
Xylidine
0.03
cáÖK=OK Effect of temperature and pH on VAO activity.
alcohol oxidase having molecular mass 71~83 kDa [20]. The native enzyme showed maxima at 340 nm. The purified VAO enzyme showed different spectral properties than known VAO (peak at 385 and 460) and other AAO [18,20]. The purified VAO enzyme also showed different spectral properties from that of laccase (peak at 280 and 660 nm) [21]. These observations indicate the presence of different type of oxidative enzyme in Comamonas sp. UVS. bÑÑÉÅíë=çÑ=ée=~åÇ=qÉãéÉê~íìêÉ=çå=~å=båòóãÉ=^Åíáîáíó=
The activity of the VAO enzyme as a function of pH is shown in Fig. 2. HCl-KCl buffer (0.05 M) was used for pH 1.0~2.0. Citrate phosphate buffer (0.05 M) was used for pH 3.0~5.0 and sodium phosphate buffer (0.05 M) was used for pH 6.0~8.0. Enzyme activity exhibits a significant dependency on the pH value of the medium. With rising pH values, activity increases to a maximum (pH optimum) and drops to almost zero in the alkaline region. Optimum pH value for VAO was 3.0. The optimum pH of 5.0 for AAO was reported by other workers [19,20]. The enzyme lost its activity after 5 h and 15 h at pH 3.0 and pH 7.0, respectively. The optimum temperature of VAO enzyme was determined at pH 3.0. The maximum activity was observed at 65°C (Fig. 2). Enzymatic activity declined with an increase or decrease in temperature. The optimum 55°C temperature for AAO was also reported by Guillen et al. [20]. In contrast to our results various optimum temperatures for AAO activity were reported by other workers [19]. pìÄëíê~íÉ=péÉÅáÑáÅáíó=
Substrate specificity was determined at the optimum pH for each substrate. As shown in Table 2, eight different substrates were used to study substrate specificity of VAO. Among various substrates used this enzyme showed maximum activity with n-propanol.
cáÖK=PK Lineweaver-Burk plot of enzyme kinetics.
háåÉíáÅ=píìÇáÉë=çÑ=íÜÉ=s^l=båòóãÉ=
The effects of increasing concentrations of the substrate (veratryl alcohol) on the activity of purified enzyme have been studied. Km and Vmax values for the purified VAO enzyme, using veratryl alcohol as substrate was determined at optimal conditions. The Km and Vmax values obtained were 2.50 mM and 0.45 μM/min, respectively (Fig. 3). bÑÑÉÅíë=çÑ=jÉí~ä=fçåë=~åÇ=fåÜáÄáíçêë=çå=íÜÉ=^Åíáîáíó=çÑ= s^l=båòóãÉ=
The effects of metal ions and inhibitors on the VAO activity were tested by using veratryl alcohol as substrate (Table 3). VAO enzyme was completely inhibited by 0.5 mM sodium azide. L-cysteine, dithiothreitol, and the metal chelator, EDTA had only a slight inhibitory effect. Many substances may alter the activity of an enzyme by influencing the binding of substrate and/or its turnover number. Substances that reduce an enzyme’s activity in this way are now known as inhibitors. Many inhibitors have structural resemblance with their enzymes substrate. Such inhibitors are commonly used to probe the chemical and conformational nature of a substrate-binding site as part of an effort to elucidate the enzyme’s catalytic mechanism. Sodium azide’s toxicity toward an enzyme is mainly due to its strong coordination ability with the metal within the active site, which provokes changes
Biotechnol. Bioprocess Eng. PTP=
q~ÄäÉ=PK= Effects of inhibitors and metal ions on VAO activity Inhibitor/Metal ions
Relative activity (%) at 0.5 mM concentration
None
100
Sodium azide
0
L-cysteine
70
EDTA
75
Dithiothreitol
85
BaCl2
100
CaCl2
100
NaCl
100
MgCl2
90
KCl
75
MnCl2
62
CdCl2
65
HgCl2
52
ZnCl2
35
CuCl2
15
cáÖK=RK UV-Vis spectral scans containing Red HE7B dye and decolorized sample of Red HE7B by VAO enzyme.
Effect of pH on dye decolorization using VAO is shown in Fig. 4. Although the optimum pH of VAO was 3.0, both the dyes decolorized maximally at pH 7.0. This might be due to increased stability of enzyme at neutral pH. The pH optima is also depends on the structure of the substrate. The enzyme lost its activity after 5 h and 15 h at pH 3.0 and pH 7.0, respectively. We have checked the decolorization of dyes after 5 h and 15 h, respectively. In contrast to our results, some workers have showed that the degradation of industrially important dyes by HRP [11,26], manganese peroxidase [27], and laccase [28,29] was also more in the buffer of lower pH values. Purified LiP from A. calcoaceticus degraded 10 different dyes at various extents tested from different classes at neutral pH, indicating that enzyme contributes extensive decolorization ability at neutral pH [30]. rsJsáëáÄäÉ=péÉÅíê~=çÑ=oÉÇ=ebT_=~åÇ=aáêÉÅí=_äìÉ=dii= cáÖK=QK Effect of pH on decolorization of RRHE7B and DBGLL.
in the coordination number and conformation of the active site. The reaction between the copper amine oxidase and azide probably hinders the bond of the precursor tyrosine to the copper. This prevents the formation of this key intermediate and inhibits the activity of the oxidase [22]. L-Cysteine can act as a reducing agent [23]. The enzyme was inhibited by 0.5 mM Cu2+ (85%), Zn2+ (65%), Hg2+ (48%), and Cd2+ (35%) (Table 3). aÉÅçäçêáò~íáçå=çÑ=qÉñíáäÉ=aóÉë=
Decolorization of the textile dyes by oxidative enzymes was reported earlier in literature [24,25]. Purified VAO can degrade two textile dyes (40 μg/mL each) namely Red HE7B (57.5%) and Direct Blue GLL (51.09%) within 15 h.
Several investigators have earlier shown that the aromatic compounds were get degraded or precipitated by the action of peroxidases/polyphenol oxidases [4,5,26,31,32]. The absorption spectra for the treated dyes exhibited decrease in the absorbance at various wavelengths in their spectral regions as compared to the untreated dyes (Figs. 5 and 6). No precipitate formation was observed during the VAO treatment with dyes. These results suggested that the decrease in the absorbance was due to degradation of dye by the VAO enzyme. aÉÖê~Ç~íáçå=^å~äóëáë
HPLC analysis of extracted sample showed original dye Red HE7B at a retention time 1.82 min (Fig. 7A). The peaks for metabolites were observed at retention time 2.620, 2.871, and 5.845 min after complete degradation (Fig. 7B). The different degradation pattern of Red HE7B was reported by Kalme et al. [33]. They showed three major metabolites at
PTQ=
^=
_=
cáÖK=SK UV-Vis spectral scans containing Direct Blue GLL dye and decolorized sample of Direct Blue GLL by VAO enzyme.
^=
cáÖK=UK HPLC profile of control Direct Blue GLL (A) and its degraded products extracted after decolorization by VAO enzyme (B).
_=
cáÖK=TK HPLC profile of control Red HE7B (A) and its degraded products extracted after decolorization by VAO enzyme (B).
retention times 1.94, 2.16, and 2.54 min after degradation of Red HE7B by Pseudomonas desmolyticum NCIM 2112. HPLC chromatogram of Direct Blue GLL showed original dye at a retention time 1.560 min (Fig. 8A). The peaks for metabolites were observed at retention time 2.648, 2.886, 3.113, 3.653, 3.892, 4.981, and 5.648 min after complete degradation (Fig. 8B). GC-MS analysis was carried out to investigate the metabolites formed during the degradation process. Microorganisms metabolise recalcistrant compounds with the help of their enzymes such as peroxidases
cáÖK=VK Proposed pathway for degradation of Red HE7B by VAO purified from `çã~ãçå~ë sp. UVS. The compound represented by alphabet in bracket has not been found, but their existence is rationalized as necessary intermediates for the final products found. The compound represented by Arabic number in bracket has been found in reaction mixture.
and laccases. A pathway has been proposed for degradation of Red HE7B and Direct Blue GLL by VAO (Figs. 9 and 10) Azo dyes can be degraded by oxidative cleavage [6]. Oxidative.
Biotechnol. Bioprocess Eng. PTR=
showed different characteristics than reported VAO. Studies also confirmed an involvement of VAO in the oxidative cleavage and desulphonation of the dyes Red HE7B and Direct Blue GLL. The oxidative cleavage avoids formation of carcinogenic amines. As VAO is effective in the degradation of dyes, it can be used as an efficient tool for the detoxification of dye wastes. Received December 29, 2008; accepted January 28, 2009
REFERENCES
cáÖK=NMK Proposed pathway for degradation of Direct Blue GLL by VAO purified from `çã~ãçå~ë sp. UVS. The compound represented by alphabet in bracket has not been found, but their existence is rationalized as necessary intermediates for the final products found. The compound represented by Arabic number in bracket has been found in reaction mixture.
cleavage of Red HE7B yielded an unknown product. This product further undergoes desulfonation to give naphthalene1,2,5-triol. Oxidative cleavage of Direct Blue GLL gave an unidentified product. The desulfonation of this product yielded naphthalene-1,4,6-triol. The structure of detected compounds was assigned from the m/z value obtained. Kalme et al. [33] obtained different metabolites other than naphthalene-1,2,5triol during decolorization of Red HE7B. The azo linkage is susceptible to reduction, which generates potentially carcinogenic aromatic amines [6]. Oxidative cleavage plays an important role in detoxification of azo dyes because this reaction releases azo linkages as molecular nitrogen, which prohibits aromatic amine formation. Oxidative biodegradation of azo dyes primarily involved the ligninolytic fungus P. chrysosporium as the decolorizing microorganism and implicated its lignin degrading enzyme system in the decolorization process. Bacterial extracellular azo dye oxidizing peroxidases have been characterized in Streptomyces chromofuscus [34]. However, peroxidases played vital role in dye decolorization [30].
CONCLUSION Veratryl alcohol oxidase from Comamonas sp. strain UVS
1. McMullan, G., C. Meehan, A. Conneely, N. Kirby, T. Robinson, P. Nigam, I. Banat, R. Marchant, and W. Smyth (2001) Microbial decolorization and degradation of textile dyes. Appl. Microbiol. Biotechnol. 56: 81-87. 2. Slokar, Y. and L. Majcen (1998) Methods of decoloration of textile wastewaters. Dyes Pigments. 37: 335-356. 3. Cripps, C., J. Bumpus, and S. Aust (1990) Biodegradation of azo and heterocyclic dyes by Phanerochaete chrysosporium. Appl. Environ. Microbiol. 56:1114-1118. 4. Husain, Q. and U. Jan (2000) Detoxification of phenols and aromatic amines from polluted wastewater by using phenol oxidases. J. Sci. Ind. Res. 59: 286-293. 5. Duran, N. and E. Esposito (2000) Potential applications of oxidative enzymes and phenoloxidase-like compounds in wastewater and soil treatment. Appl. Cataly. B: Environ. 28: 83-99. 6. Chivukula, M. and V. Renganathan (1995) Phenolic azo dye oxidation by laccase from Pyricularia oryzae. Appl. Environ. Microbiol. 61: 4374-4377. 7. Scheibner, K., M. Hofrichter, and W. Fritsche (1997) Mineralization of 2-amino-4,6-dinitrotoluene by manganese peroxidase of the white-rot fungus Nematoloma frowardii. Biotechnol. Lett. 19: 835-839. 8. Heinfling, A., J. Martinez, A. Martinez, M. Bergbauer, and U. Szewzeyk (1998) Purification and characterization of peroxidase from the dye-decolorizing fungus Bjerkandera adusta. FEMS Microbiol. Lett. 165: 43-50. 9. Van Aken, B., M. Hofrichter, K. Scheibner, A. Hatakka, H. Naveau, and S. Agathos (1999) Transformation and mineralization of 2,4,6-trinitrotoluene (TNT) by manganese peroxidase from the white-rot basidiomycete Phlebia radiata. Biodegradation 10: 83-91. 10. Nie, G., N. Reading, and S. Aust (1999) Relative stability of recombinant versus native peroxidases from Phanerochaete chrysosporium. Arch. Biochem. Biophy. 34: 365 -328. 11. Bhunia, A., S. Durani, and P. Wangikar (2001) Horse radish peroxidase catalyzed degradation of industrially important dyes. Biotechnol. Bioeng. J. 72: 562-567. 12. Shaffiqu, T., J. Roy, R. Nair, and T. Abraham (2002) Degradation of textile dyes mediated by plant peroxidases. Appl. Biochem. Biotechnol. 102: 315-326. 13. Ward, G., Y. Hadar, I. Bilkis, L. Konstantinovsky, and C. Dosoretz (2001) Initial steps of ferulic acid polymeriza-
PTS=
14.
15. 16.
17.
18. 19.
20.
21.
22.
23. 24.
tion by lignin peroxidase. J. Biol. Chem. 276: 1873418741. Ward, G., Y. Hadar, and C. Dosoretz (2001) Inactivation of lignin peroxidase during oxidation of the highly reactive substrate ferulic acid. Enzyme Microb. Technol. 29: 34-41. Ander, P. and L. Marzullo (1997) Sugar oxidoreductases and veratrole oxidase as related to lignin degradation. J. Biotechnol. 53: 115-131. Jadhav, U., V. Dawkar, G. Ghodake, and S. Govindwar (2008) Biodegradation of Direct Red 5B, textiles dye by newly isolated Comamonas sp. UVS. J. Hazard. Mater. 158: 507-516. Jadhav, U., V. Dawkar, A. Telke, and S. Govindwar (2008) Decolorization of Direct Blue GLL with enhanced lignin peroxidase enzyme production in Comamonas sp. UVS. J. Chem. Technol. Biotechnol. 84: 126-132. Bourbonnais, R. and M. Paice (1988) Veratrole oxidases from the lignin-degrading basidiomycete Pleurotus sajor-caju. Biochem. J. 255: 445-450. Kim, S., N. Suzuki, Y. Uematsu, and M. Shoda (2001) Characterization of aryl alcohol oxidase produced by dye-decolorizing fungus, Geotrichum candidum Dec l. J. Biosci. Bioeng. 91: 166-172. Guillen, F., A. Martinez, and M. Martinez (1992) Substrate specificity and properties of the aryl-alcohol oxidase from the ligninolytic fungus Pleurotus eryngii. Eur. J. Biochem. 209: 603-611. Kiiskinen, L., L. Viikari, and K. Kruus (2002) Purification and characterization of a novel laccase from the ascomycete Melanocarpus albomyces. Appl. Microbiol. Biotechnol. 59: 198-204. Schwartz, B., A. Olgin, and J. Klinman (2001) The role of cooper in topa quinone biogenesis and catalysis, as probed by azide inhibiton of a copper amine oxidase from yeast. Biochemistry 40: 2954-2963. Wesche-Ebeling, P. and M. Montgomery (1990) Strawberry polyphenol oxidase: extraction and partial characterization. J. Food Sci. 55: 1320-1325. Nagal, M., T. Sato, H. Watanabe, K. Saito, M. Kawata, and H. Enei (2002) Purification and characterization of
25.
26.
27.
28. 29.
30.
31. 32. 33.
34.
an extraceluular laccase from the edible mushroom Lentinula edodes and decolorization of chemically different dyes. Appl. Microbiol. Biotechnol. 60: 327-335. Baldrian, P. (2004) Purification and characterization of laccase from the white rot fungus Daedalea quercina and decolorization of synthetic dyes by the enzyme. Appl. Microbiol. Biotechnol. 63: 560-563. Mohan, S., K. Prasad, N. Rao, and P. Sarma (2005) Acid azo dye degradation by free and immobilized horseradish peroxidase (HRP) catalyzed process. Chemosphere 58: 1097-1105. Christian, V., R. Shrivastava, C. Novotny, and B. Vyas (2003) Decolorization of sulfonaphthalene dyes by manganese peroxidase activity of the white-rot fungus Phanerochaete chrysosporium. Folia Microbiol. 48: 771 -774. Palmieri, G., P. Giardina, and G. Sannia (2005) Laccasemediated Remazol Brilliant Blue R decolorization in a fixed-bed bioreactor. Biotechnol. Prog. 21: 1436-1441. Camarero, S., D. Ibarra, M. Martinez, and A. Martinez (2005) Lignin derived compounds as efficient laccase mediators for decolorization of different types of recalcitrant dyes. Appl. Environ. Microbiol. 71: 1775-1784. Ghodake, G., S. Kalme, J. Jadhav, S. Govindwar (2008) Purification and partial characterization of lignin peroxidase from Acinetobacter calcoaceticus NCIM 2890 and its application in decolorization of textile dyes. Appl. Biochem. Biotechnol. 152: 6-14. Tatsumi, K., S. Wada, and H. Ichikawa (1996) Removal of chlorophenols from wastewater by immobilized horse radish peroxidase. Biotechnol. Bioeng. J. 51: 126-130. Mielgo, I., M. Moreira, and G. Feyoo (2001) A packed bed fungal bioreactor for the continuous decolorization of azo dye (Orange II). J. Biotechnol. 89: 99-106. Kalme, S., G. Ghodake, and S. Govindwar (2007) Red HE7B degradation using desulfonation by Pseudomonas desmolyticum NCIM 2112. Int. Biodet. Biodeg. 60: 327333. Pasti-Grigsby, M. B., N. S. Burke, S. Goszczynski, and D. L. Crawford (1996) Transformation of azo dye isomers by Streptomyces chromofuscus A11. Appl. Environ. Microbiol. 62: 1814-1817.
