Laccase Production and Enzymatic Modification of Lignin by a Novel Peniophora sp

Laccase Production and Enzymatic Modification of Ligninby a Novel Peniophora sp.

Shiv Shankar & Shikha

Received: 8 October 2011 /Accepted: 11 December 2011 /Published online: 28 December 2011# Springer Science+Business Media, LLC 2011

Abstract A novel laccase producing Basidiomycete Peniophora sp. (NFCCI-2131) wasisolated from pulp and paper mill effluent. The optimal temperature and initial pH forlaccase production by the isolate in submerged culture were found to be 30 and 4.6°C,respectively. Maltose (20 gl−1) and tryptone (1.0 gl−1) were the most suitable carbon andnitrogen sources for laccase production. Cu2+ (1.0 mM) and veratryl alcohol inducedmaximum laccase production giving 6.6 and 6.07 U/ml laccase activity, respectively. Underoptimised culture conditions, 7.6 U/ml activity was obtained, which was 2.4 times higherthan that was achieved in basal medium. An evaluation of the delignification efficiency ofthe crude enzyme in the presence of redox mediators [2,2’-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) and (1-hydroxybenzotriazole)] revealed structural changes in lignin andexistence of many active centres for both chemical and biological degradation of ligninfollowing enzymatic treatment.

Keywords Peniophora sp. . Laccase . Lignin . Delignification . FTIR

AbbreviationsMEA Malt extract agarFTIR Fourier transform infrared spectrometerABTS 2,2’-Azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)HBT 1-HydroxybenzotriazoleNFCCI National Fungal Culture Collection of IndiaARI Agharkar Research Institute

Introduction

Laccases (benzenediol:oxygen oxidoreductase, E.C 1.10.3.2) are multi-copper oxidaseswidely distributed among plants, insects and fungi [1]. Laccases are considered as eco-

Appl Biochem Biotechnol (2012) 166:1082–1094DOI 10.1007/s12010-011-9496-4

S. Shankar : Shikha (*)Department of Environmental Science, Babasaheb Bhimrao Ambedkar University(A Central University), Vidya vihar, Rae bareli Road, Lucknow 226025, Uttar Pradesh, Indiae-mail: [email protected]

friendly enzymes because of their ability to catalyse the oxidation of a phenolic substrate bycoupling it to the reduction of O2 to water, without resulting in the formation of harmfulintermediate compounds [2]. The ability of laccases to oxidise a wide variety of substratesmake them potential candidate for several biotechnological and environmental applications[3], e.g., elimination of toxic compounds from polluted effluents through oxidative enzy-matic coupling and precipitation of the contaminants [4], as biosensors for phenols [5], indelignification, demethylation and bleaching of kraft pulp [6–8]. In addition, laccases mayalso act on chromophore compounds, suggesting their extended application in industrialdecolourisation processes as well [9, 10].

In pulp and paper industry, removal of lignin from wood is required during papermanufacturing process [11]. Lignin is a complex three-dimensional polymer present in allwoody plants and different lignocellulosic residues, generally used as raw material for papermanufacturing. It contributes structural integrity to plant cell wall owing to its linkages withcellulose and hemicellulose residues [12, 13]. The brown colour of pulp is due to thepresence of lignin; removal of which is indispensable during the process of paper makingfor aesthetic reasons. Removal of this lignin by incorporating enzymes is considered asdelignification coupled with biobleaching and is an environment friendly process as comparedto conventional bleaching [14].

Laccases play a significant role in the biodegradation of lignin [6]. They have beenspecifically proved to oxidise lignin and other model compounds in the presence of redoxmediators of low molecular weight. Such ‘laccase mediator system’ is known to boost theaction of the enzyme. 1-Hydroxybenzotriazole (HBT) and 2,2’-azino-bis(3-ethylbenzthiazo-line-6-sulphonic acid) (ABTS) are two most widely used mediators for this purpose [15].

White rot fungi are effective lignin degraders because of their ability to produce extra-cellular laccases which are highly effective in degrading lignin [16]. In order to utiliselaccases more efficiently, it is necessary to have a better understanding about the propertiesof this industrially important enzyme both at a molecular and kinetic level as well. For allindustrial applications, large amounts of crude and purified laccases are required [1].However, in most of the reported fungi, the yield of laccases is too low even when theirgenes are expressed in various heterologous hosts [17]. Hence, there is a need to explore newstrategies for increased production of laccases which may include isolation and screening ofnovel fungal strains, improvement in enzyme production by optimising media conditionsand understanding the structural modifications in lignin imparted by the enzyme.

The present study is an evaluation of the laccase production efficiency of the isolatedfungal strain, a novel Peniophora sp., optimization of the fermentation parameters favouringhigh laccase yield under submerged culture conditions, and a study of the influence oflaccase mediator system in delignification of kraft pulp, through Fourier transform infraredspectrometer (FTIR), which is likely to provide an insight into the laccase-induced structuralchanges in lignin.

Materials and Methods

Isolation and Screening of Microorganisms

Malt extract agar medium (MEA) containing (in grams per liter) malt extract, 20; peptone,1.0; and glucose 20 was used for isolation, screening and enzyme production. For isolation,diverse environmental samples were taken from various ecological niches, like decayingwood and tree barks from the campus of Babasaheb Bhimrao Ambedkar University,

Appl Biochem Biotechnol (2012) 166:1082–1094 1083

Lucknow, U.P., India, pulp and paper mill effluent (Yash Paper Mills, Faizabad, UttarPradesh, India; Century Pulp and Paper Mill, Lalkuan, Nainital, Uttarakhand, India) as wellas rhizospheric soil. The samples were diluted and spread on MEA agar plates containing5.0 mM Guaiacol [18]. The plates were incubated at 37°C, pH 4.6 for 96 h. One of thepromising isolate was sent to Agarkar Research Institute (ARI), Pune, India for identifica-tion, where it was identified as a novel Peniophora sp. and was granted an accession numberNFCCI-2131 by National Fungal Culture Collection of India.

Inoculum Preparation and Cultivation Conditions

Fungal inoculum was prepared by growing the strain under static condition at 30 C andpH 4.6 in 500 ml Erlenmeyer flasks containing 100 ml of the standard malt extractbroth. Subsequent to 7 days of cultivation, the mycelial homogenate (1.0 ml) was usedto inoculate 500 ml flask containing 100 ml of the standard malt extract broth. Theinitial pH of the medium was adjusted to 4.6 prior to sterilisation. The inoculated flaskswere incubated in an incubator for 28 days at 30°C under static condition. At regulartime intervals (48 h), laccase activity was assayed in the extracellular fluid obtainedsubsequent to the separation of solids by filtration, followed by centrifugation(10,000×g, 10 min) at 4°C.

Laccase Assay

Laccase (EC 1.10.3.2) activity was determined by monitoring the change in absorbance at465 nm related to the rate of oxidation of 5 mM guaiacol to its corresponding radical in50 mM sodium acetate buffer (pH 4.6) at 37°C [19], against an appropriate blank.

Optimization for Laccase Production Under Submerged Fermentation

The optimization studies included the production of laccase under different pH (3.6–8.6) andincubation temperature (20–40 C) of the growth medium, supplementation of the growthmedium with different carbon sources (20 gl−1), such as glucose, maltose, D-manitol, starch,

D-ribose, D-sorbitol and fructose as well as by different organic and inorganic nitrogensources (1.0 gl−1), such as ammonium nitrate, calcium nitrate, sodium nitrite, ammoniumacetate, ammonium carbonate, nicotinic acid, potassium nitrate and L-histidine, L-asparagine,urea, L-aspartic acid, glycine, L-arginine and yeast extract, respectively.

Effect of Inducers and Copper Sulphate on Laccase Production

In order to study the effect of inducers on enzyme production, various aromatic com-pounds (2,5-xylidine, vanillin, phloroglucinol, H-hydroxybenzoic acid, o-dianisidine andveratryl alcohol) were added to the cultivation medium at a concentration of 1 mM at thetime of inoculum transfer. All the inducers except vanillin were dissolved in ethanol whilevanillin was dissolved in deionized water. The final concentration of ethanol in the growthmedium was always less than 0.5% and an equivalent amount of ethanol was added tocontrol flasks without an aromatic inducer. The production of laccase is very oftenenhanced by heavy metal ions, especially copper. To ascertain the possible effect ofcopper sulphate on extracellular laccase production, copper sulphate was added at varyingconcentrations (0.5–2.0 mM) to the cultivation medium prior to inoculation with fungalsuspension.

1084 Appl Biochem Biotechnol (2012) 166:1082–1094

Enzymatic Treatment of Pulp

The capability of the laccase to delignify kraft pulp in solo as well as in the presence andabsence of mediators was evaluated by treating the pulp with an enzyme alone (5.0 U/ml) aswell as with an enzyme in the presence of ABTS (1 mM) and HBT (1 mM) for 24–72 h. Allthe experiments were performed using 1.0 g of pulp, dried at 50°C and pH 4.6.

FTIR of Pulp

Residual lignin from treated and untreated pulp with and without mediators (ABTS andHBT) was analysed by FTIR spectroscopy [20]. Following an incubation of 24, 48 and 72 h,lignin samples were embedded in KBr disc and spectra were recorded with spectrometer(Perkin Elmer-1600 series, USA) at room temperature. The FTIR database for lignin wastaken as a reference for assignment of absorbance peaks [21].

Results and Discussion

The laccase-producing fungi were isolated from various environmental samples following achromogenic screening method using solid media containing indicator compound guaiacol.Out of the 15 fungal isolates, six were laccase positive. From the laccase-positive fungi, onepotential candidate FS-SH was selected for further studies on the basis of size of the reddishbrown zone in the medium formed due to oxidative polymerization of guaiacol which washighest as compared to other isolated strains. The isolated strain was identified as a novelPeniophora sp. by ARI, the same being deposited at NFCCI.

It has been well documented that microbial laccase production is affected by cultureconditions, such as pH, temperature, carbon and nitrogen sources and the presence ofinducers to variable extent [22]. Laccase production by Peniophora sp. was monitoredunder submerged fermentation at different pH values (pH 3.6 to 8.6).

Results (Fig. 1) revealed an increase in laccase production from pH 3.6 to 4.6, butthereafter, laccase activity decreased. The maximum laccase activity (3.09 U/ml) as recordedon 22nd day of incubation was obtained at pH 4.6. However, the laccase activity at pH 5.6was approximately 59.23% more than the activity as observed at pH 3.6. Poor mycelialgrowth at elevated pH may be one of the reasons restricting laccase yield [23]. Variousresearchers have also reported optimum laccase production in the culture medium having pHbetween 4 and 6. [24].

The effect of different incubation temperatures (20–40°C) was studied over a period of26 days of incubation. Results (Fig. 2) showed that with an increase in incubation temper-ature beyond 30°C, laccase activity decreased. A temperature of 30°C was observed to giveoptimal laccase activity throughout the incubation period. Our results are in conformity withVasdev et al. [25], where it was reported that temperatures higher than 30°C reduce theactivity of lignolytic enzymes.

Seven different carbon sources were tested for laccase production. The results (Fig. 3)showed that the maximum laccase activity (3.5 U/ml) was induced by maltose. A 3.09 U/mllaccase activity was obtained with glucose (control) which was 13% lower than maltose.Comparatively lower activities were shown by mannitol, fructose followed by D-ribose, D-sorbitol and starch. Similar results have also been obtained in a study conducted by Wang[26] where maltose as a carbon source has been found to support highest laccase productionthan any other carbon sources.


The effect of different organic and inorganic nitrogen sources were studied on laccaseproduction in submerged medium (Fig. 4a, b). Among the organic sources, tryptone (5.13 U/ml) was the best nitrogen source giving approximately 1.68-fold higher activity than peptone(3.05 U/ml). Tryptone has also been reported to strongly improve laccase production as anorganic nitrogen source by Dong et al. [27].

Apart from tryptone, nicotinic acids as well as L-aspartic acid were also found to stimulatelaccase yield marginally, as compared to peptone (control). However, rest of the organicnitrogen sources did not seem to stimulate laccase production (Fig. 4a).

0

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0 12 14 16 18 20 22 24 26Incubation period (days)

Lac

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/ml)

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25

30

35

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Fig. 2 Laccase production by Peniophora sp. at different temperatures. An average of three observations.Standard error±2.75%; p<0.002. The p value refers to the comparison of laccase activity at 30 C with activityat different temperatures. All the comparisons are statistically significant. Carbon and nitrogen sources usedare glucose (−2%) and peptone (−0.1%)

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/ml)

pH 3.6

pH 4.6

pH 5.6

pH 6.6

pH 7.6

pH 8.6

Fig. 1 Effect of pH on laccase production by Peniophora sp. An average of three observations. Standarderror±3.95%; p<0.002. The p value refers to the comparison of laccase activity at pH 4.6 with activity atdifferent pHs. All the comparisons are statistically significant. Carbon and nitrogen sources used are glucose(−2%) and peptone (−0.1%)


Among inorganic nitrogen sources tested (Fig. 4b), ammonium acetate appeared to be thebest nitrogen sources giving 4.63 U/ml activity, followed by ammonium sulphate (4.44 U/ml),as compared to the 3.05 U/ml activity in the control (devoid of inorganic nitrogen source).Wang et al. [26] also reported ammonium acetate as an efficient inorganic nitrogen sourcesupporting highest laccase yield [26].

Since the inducing effect of Cu2+ on laccase production has been reported in variousstudies, the effect of copper sulphate was tested for laccase production by Peniophora sp.CuSO4 (1.0 mM) was found to support maximum laccase production (6.6 U/ml) as recordedon the 22nd day of incubation (Fig. 5) which was approximately 2.13-fold more than thecontrol (medium without CuSO4). Copper requirement by microorganisms are generallysatisfied by very low concentration [28]. In our study, 1.0 mM of copper sulphate was foundto induce maximum laccase production which is in sharp contrast with the concentrationstypically used in cultivation media (2–600 μM) as reported for the production of laccase ineither wild type or recombinant strains [29, 30]. However, our findings are in corroborationwith some researchers who have demonstrated that 1–2 mM Cu2+ may increase the enzymeactivity in some Trametes and Pleurotus species [31, 32]. Niladevi and Prema [33] alsoobtained maximum laccase activity when CuSO4 was used at a concentration of 1.0 mM. Itcan be suggested that Cu2+ ions may form an integral prosthetic group and thereby mayresult in an increase in laccase production [34].

Apart from Cu2+, aromatic and phenolic compounds have been widely used as inducersfor laccase production [35]. The nature of the compound that induces laccase activity differsgreatly with the species. Effect of six different inducers was studied on the production oflaccase by Peniophora sp. The results revealed that all the inducers (Fig. 6) enhanced thelaccase production to variable extent, except for O-dianisidine, which suppressed the laccaseproduction giving only 1.4 U/ml laccase activity which was lower than the control (3.05 U/ml). Veratryl alcohol resulted in a substantial increase in laccase yield, (6.07 U/ml) asobserved on 20th day of incubation. H-hydroxybenzoic acid was also found to supportsignificant laccase production giving 5.59 U/ml activity, followed by 2,5-xylidine, vanillinand phloroglucinol (4.47, 3.92 and 3.4 U/ml, respectively). The results obtained in presentstudy coincide with the results obtained by Lee et al. [36] where veratryl alcohol wasreported to induce substantial laccase production of laccase. The aromatic inducers in thepresent study were having structural similarity to either lignin or polyphenols; most of whichhave been reported as effective laccase inducers in fungal strains [37].

Subsequent to the optimization of different fermentation parameters, laccase productionwas determined in the optimised medium by combining the optimum levels obtained for

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

Glucose (Control)

Maltose

Fructose

D-manitol

Starch

D-Ribose

D-Sorbitol

Laccase activity (U/ml)

Car

bon

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Fig. 3 Effect of carbon sourceson laccase production by thePeniophora sp. at pH 4.6 andtemperature 30°C. Averageof three independent experiments.Nitrogen source: peptone 0.1%;p value <0.002. The p value refersto the comparison of laccaseactivity at 2% glucose with othercarbon sources. All the compari-sons are statistically significant


individual parameters under submerged cultivation. Results (Fig.7) revealed that laccaseproduction in the finally optimised medium (carbon source, maltose 20 gl−1; nitrogensource, tryptone 1.0 gl−1; CuSO4, 1.0 mM; veratryl alcohol, 1.0 mM; pH 4.6; and

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Fig. 4 a Effect of organic nitrogen sources on laccase production by Peniophora sp. at pH 4.6 and temperature30°C. Average of three independent experiments. Carbon source used: glucose 2%; p value <0.002. The p valuerefers to the comparison of laccase activity at 0.1 % nitrogen (peptone) with other nitrogen sources. All thecomparisons are statistically significant. b Effect of inorganic nitrogen sources on laccase production byPeniophora sp. at pH 4.6 and temperature 30°C. Average of three independent experiments. Carbon source used:glucose 2%; p value <0.002. The p value refers to the comparison of laccase activity at 0.1% nitrogen (peptone)with inorganic nitrogen sources. All the comparisons are statistically significant


temperature, 30°C) was 7.6 U/ml which was approximately 2.49-fold higher than that wasobtained in the basal medium (3.05 U/ml).

The structure of lignin is very complex and irregular and is comprised of aromatic ringswhich are connected with functional groups such as hydroxyl, carbonyl, methoxy andcarboxylic groups [38]. The FTIR spectra of pulp with enzyme and mediators like ABTSand HBT reflect its effect in terms of changes in band positions and disappearance andreappearance of new bands.

Generally, lignin IR spectrum has a strong band between 3,500 and 3,100 cm−1 assignedto OH stretching vibrations, caused by presence of alcoholic and phenolic hydroxyl groupsinvolved in hydrogen bonding [39]. Our results displayed a strong band between 3,440 and3,445 cm−1 which invariably remained intact irrespective of enzymatic treatment either withor without mediators (Figs. 8, 9 and 10). However, the intensity of bands was found todecrease subsequent to the treatment with enzyme which was further reduced under laccase–

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0.5 mM

1.0 mM

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2.0 mM

Fig. 5 Effect of different concentration of CuSO4 on laccase production by Peniophora sp. at pH 4.6, temper-ature 30 C, inMEmedium. Average of three independent experiments. Standard error±4.5%; p value <0.002. Thep value refers to the comparison of laccase activity without CuSO4 with concentrations of CuSO4. All theexperiments are statistically significant

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Xylidine

Vanillin

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Veratryl Alcohol

Fig. 6 Effect of inducers on lac-case production by Peniophorasp. at pH 4.6 and temperature,30°C. An average of three obser-vations. Standard error±2.66%;p<0.002. The p value refers to thecomparisons of laccase activitywithout any inducer with differentphenolic inducers. All the com-parisons statistically significant


ABTS and HBT treatments. This may be attributed to the methylation of O–H bonds whichundergoes splitting, H being replaced by CH3 groups resulting in a decrease in the amount ofOH groups and hence the intensity. The spectra of both treated and untreated lignin under allconditions revealed a band between 2,917 and 2,933 cm−1 (Fig. 8, 9 and 10) which may beattributed to C–H stretching vibrations of the methoxyl group [21, 40].

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Fig. 7 Enzyme production byPeniophora sp. in optimised andbasal medium; p<0.002. The pvalue refers to the comparison oflaccase activity in basal mediumwith optimised medium. All thecomparisons are statisticallysignificant

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5004000 3500 3000 2500 2000 1500 1000

Fig. 8 FTIR spectra of sugarcane pulp treated with crude laccase for different time intervals: a Untreated pulp(control); b Pulp treated with crude laccase, 24 h; c Pulp treated with crude laccase, 48 h; d Pulp treated withcrude laccase, 72 h


An absorption band between 1,630 and 1,660 cm−1 was also located under all conditionswhich may be C0C vibrations of aromatic and polyaromatic rings (1,630–1,680 cm−1) [38].Similarly, bending vibrations between 1,374 and 1,383 cm−1 under all conditions emanatedphenolic hydroxyls and symmetric deformation vibrations of C–H in methoxyl group(1,350–1470 cm−1) [21, 41].

The band at 1,086 cm−1 present in untreated pulp might be caused by the deformation of C–Obands in secondary alcohol and aliphatic ethers [42]. Interestingly, this band disappearedsubsequent to the enzymatic treatment both without and with mediators. However, appearanceof new bands at 1,061 and 1,063 cm−1 following 24 and 72 h of pulp incubation with enzyme,without mediators revealed the presence of primary alcohol (1,040–1,061 cm−1) [38], while theappearance of a new band at 1,163 cm−1 after 24 h of incubation may be attributed to theformation of tertiary alcohol (1,150–1,200 cm−1) [38] which disappeared subsequent to 48 h ofincubation. Subsequent to the 48 h of treatment of pulp with enzyme alone further revealed a newpeak at 1,025 cm−1 which may be attributed to C–O stretch of alcohol/phenols [40] as a result ofthe action of enzyme on aromatic ring of lignin. A peak located in untreated pulp at 615 cm−1 waslost and new bands were located between 571 and 901 cm−1 under all treatments which may beattributed to deformation vibrations of C–H bonds associated to aromatic rings [38, 43].

FTIR spectra of pulp treated with laccase ABTS system revealed three new bands at1,105, 1,113 and 1,030 cm−1 following 24 and 48 h of incubation, respectively, which may

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% T

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Fig.9 FTIR spectra of sugarcane pulp treated with crude laccase for different time intervals. a Untreated pulp(control); b pulp treated with crude laccase and HBT; 24 h; c Pulp treated with crude laccase and HBT, 48 h; dpulp treated with crude laccase and HBT, 72 h


be attributed to deformation vibrations of C–H bands in the aromatic rings [39] anddeformation vibrations of C–O bonds in primary alcohols (1,035–1,130 cm−1) [44]. How-ever, these bonds disappeared after 72 h of incubation and a new band was located at1,076 cm−1 further emanating chemical modification related to lignin degradation.

The pulp treated with laccase–HBT system emanated two new bands at 2,129 and1,417 cm−1 following 48 h of incubation. The band at 2,129 cm−1 has been reported possiblyto be caused by carbonyl group formed with highly dispersed supported copper. Since theband disappears subsequent to further incubation of 72 h, it can be attributed to low stabilityof carbonyl in Cu–CO species which can be an active centre for biological interaction oflaccases, not reported earlier. Similarly, the band at 1,417 cm−1 attributed to C–C stretch (inring) in aromatics (1,500–1,400 cm−1) [38] which too subsequently disappears following72 h of incubation. The overall results suggested modification in lignin subsequent toenzymatic treatment with and without mediators.

Conclusion

A significant difference in extracellular laccase activity was revealed in all the culturalconditions studied, as well as the finally optimised medium and the basal medium. Theisolate was also able to oxidise several phenolic substrates. In future, our endeavour shall be

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3748.10

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Fig. 10 FTIR spectra of sugarcane pulp treated with crude laccase for different time intervals. a Untreatedpulp (control); b Pulp treated with crude laccase and ABTS, 24 h; c Pulp treated with crude laccase and ABTS,48 h; d Pulp treated with crude laccase and ABTS, 72 h


to scale up the production of laccase using different agricultural/kitchen wastes and also toevaluate the biobleaching potential of the enzyme for various industrial applications. Ourisolate has been identified as a novel Peniophora sp. and this strain seems to be a prospectiveorganism for biotechnological industrial applications specially pulp and paper industry.

The fragmentation of polymeric lignin is the first requisite for lignin degradation, whichcan be subsequently metabolised intracellularly [45]. Our results revealed several structuralchanges in lignin during enzyme treatment with laccase with and without mediators asdetermined with FTIR spectroscopy. The degradation pattern observed in the present studyrevealed some of the many active centres for both chemical and biological degradation oflignin. Since the degradation of lignin is very complex, an understanding of the processcould significantly contribute towards the development of an ecologically benign technologyfor different applications including pulp and paper industry.

Acknowledgments The authors wish to thank the Council of Science and Technology, U.P., for a researchgrant to Dr. Shikha and UGC, New Delhi for research fellowship to Shiv Shankar as well as ARI, Pune, Indiafor identifying the culture.

References

1. Gianfreda, L., Xu, F., & Bollag, J. M. (1999). Bioremediation Journal, 3, 125–129.2. Kim, Y., Cho, N. S., Eom, T. J., & Shiun, W. (2002). Bulletin of the Korean Chemical Society, 23, 985–

989.3. Riva, S. (2006). Trends in Biotechnology, 24, 219–226.4. Zille, A., Munteanu, F. D., Guebitz, G. M., & Cavaco-Paulo, A. (2005). Journal of Molecular Catalysis

B: Enzymatic, 33, 23–28.5. Torrecilla, J. S., Mena, M. L., Yanez-Sedeno, P., & Garcia, J. (2008). Biochemical Engineering Journal,

38, 171–179.6. Camarero, S., Ibarra, D., Martínez, A. T., Romero, J., Gutiérrez, A., & Del Río, J. C. (2007). Enzyme and

Microbial Technology, 40, 1264–1271.7. Speranza, M., Ibarra, D., Romero, J., Martinez, A. T., Martinez, M. J., & Camarero, S. (2007).

Biocatalysis and Biotransformation, 25, 251–259.8. Shankar, S., & Shikha. (2011). Recent advances in environmental biotechnology. In P. K. Jain, V. K.

Gupta, & V. Bajpai (Eds.), Fungal laccases: production and biotechnological applications in pulp andpaper industry (pp. 73–111). Dudweller: Lap Lambert Academic Publishing Ag and Co.

9. Champagne, P. P., & Ramsay, J. A. (2007). Applied Microbiology and Biotechnology, 77, 819–823.10. Svobodova, K., Majcherczyk, A., Novotny, C., & Kuees, U. (2008). Bioresource Technology, 99, 463–471.11. Barreca, A. M., Fabbrini, M., Galli, C., Gentili, P., & Ljunggren, S. (2003). Journal of Molecular

Catalysis B: Enzymatic, 26, 105–110.12. Uffen, R. L. (1997). Journal of Industrial Microbiology and Biotechnology, 19, 1–6.13. Record, E., Asther, M., Sigoillot, C., Pages, S., Punt, P. J., & Haon, M. (2003). Applied Microbiology and

Biotechnology, 62, 349–355.14. Bajpai, P., & Bajpai, P. K. (1992). Process Biochemistry, 27, 319–325.15. Morozova, O. V., Shumakovich, G. P., Shleev, S. V., & Yaropolov, Y. I. (2007). Applied Biochemistry and

Microbiology, 43, 523–535.16. Dekker, R. F. H., Ling, K. Y., & Barbosa, A. M. (2000). Biotechnology Letters, 22, 105–108.17. Hong, Y. Z., Xiao, Y. Z., Zhou, H. M., Fang, W. M., Zhu, J., Wang, J., et al. (2006). FEMS Microbiology

Letters, 258, 96–101.18. Sundman, V., & Nase, L. (1971). Paperi ja Puu–Papper Och Tra, 2, 67–71.19. Unal, A., & Kolankaya, N. (2004). Turkish Electronic Journal of Biotechnology, 2, 17–21.20. Geng, X., & Li, K. (2000). Applied Microbiology and Biotechnology, 60, 342–346.21. Buta, J. G., Zardrazil, F., & Gallettti, G. C. (1989). Journal of Agricultural and Food Chemistry, 37,

1382–1384.22. Wesenberg, D., Kyriakides, I., & Agathos, S. N. (2003). Biotechnology Advances, 22, 161–187.


23. Sarma, P. N., Krishna, P. K., Venkata, M. S., Vijaya, B. Y., Ramanaiah, S. V., Lalit, B. V., et al. (2005).Journal of Microbiology, 43, 301–307.

24. Thurston, C. F. (2004). Microbiology, 140, 19–26.25. Vasdev, K., Dhawan, S., Kapoor, R. K., & Kuhad, R. C. (2005). Fungal Genetics and Biology, 42, 684–

693.26. Wang, J. W., Wu, J. H., Huang, W. Y., & Tang, R. X. (2006). Bioresource Technology, 97(5), 786–789.27. Dong, J. L., Zhang, Y. W., Zhang, R. H., Huang, W. Z., & Zhang, Y. Z. (2005). Journal of Basic

Microbiology, 45, 190–198.28. Labbe, S., & Thiele, D. J. (1999). Methods in Enzymology, 306, 145–153.29. Collins, P. J., & Dobson, A. D. W. (1997). Applied and Environmental Microbiology, 63, 3444–3450.30. Palmieri, G., Giardina, P., Bianco, C., Fontanella, B., & Sannia, G. (2000). Acta Biotechnologica, 20, 31–

38.31. Levin, L., Jordan, A., Forchiassin, F., & Viale, A. (2011). Revista Argentina de Microbiologia, 33, 223–

228.32. Galhaup, C., Wagner, H., Hinterstoisser, B., & Haltricha, D. (2002). Enzyme and Microbiol Technology,

30, 529–536.33. Niladevi, K. N., & Prema, P. (2008). Bioresource Technology, 99, 4583–4589.34. Rogalski, J. (2001). Journal of Basic Microbiology, 41, 185–227.35. Leonowicz, A., Cho, N. S., Luterek, J., Wilkolazka, A., Wojtas-Wasilewska, M., Matuszewska, A., et al.

(2001). Journal of Basic Microbiology, 41, 185–227.36. Lee, I. Y., Jung, K. H., Lee, C. H., & Park, Y. H. (1999). Biotechnology Letters, 21, 965–968.37. Revankar, M. S., & Lele, S. S. (2006). World Journal of Microbiology and Biotechnology, 22(9), 921–

926.38. Hergert, H. L. (1960). Journal of Organic Chemistry, 25, 405–413.39. Hergert, H. L. (1971). Infrared spectra. In K. V. Sarkanen & C. H. Ludwig (Eds.), Lignins: occurrence,

formation, structure and reactions (pp. 267–297). New York: John Willey and Sons, NY.40. Lisperguer, J., Perez, P., & Urizor, S. (2009). Journal of the Chilean Chemical Society, 54(4), 460–463.41. Khan, M. A., Idriss, Ali, K. M., & Basu, S. C. (1993). Journal of Applied Polymer Science, 49, 1547–

1551.42. Durie, R. A., Lynch, B. M., & Sternhell, S. (1960). Australian Journal of Chemistry, 13, 156–168.43. Pandey, K. K., & Nagveni, H. C. (2007). Holzals Roh-und Werkstoff, 65, 477–481.44. Bolker, N. I., & Somerville, N. G. (1963). Infrared spectroscopy of lignins. Part II. Lignins in unbleached

pulp. Pulp & Paper (Canada), 64, 187–193.45. Eriksson, K. E. L., Blanchette, R. A., & Ander, P. (1990). Biodegredation of lignin. In K. E. L., Eriksson,

R. A. Blanchette, & P. Ander (Eds.),Microbial and enzymatic degredation of wood and wood components(pp 89–180). Berlin: Springer Verlag.


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