Research Article Isolation and Physicochemical ...downloads.hindawi.com/journals/bmri/2016/3238909.pdfResearch Article Isolation and Physicochemical Characterization of Laccase from

Research ArticleIsolation and Physicochemical Characterization ofLaccase from Ganoderma lucidum-CDBT1 Isolated fromIts Native Habitat in Nepal

Prabin Shrestha1 Bishnu Joshi1 Jarina Joshi1

Rajani Malla1 and Lakshmaiah Sreerama123

1Central Department of Biotechnology Tribhuvan University Kirtipur Nepal2Department of Chemistry and Earth Sciences Qatar University Doha Qatar3Department of Chemistry and Biochemistry St Cloud State University St Cloud MN 56301 USA

Correspondence should be addressed to Lakshmaiah Sreerama lsreeramagmailcom

Received 11 June 2016 Revised 25 August 2016 Accepted 21 September 2016

Academic Editor Juan M Bolivar

Copyright copy 2016 Prabin Shrestha et alThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

At present few organisms are known to and capable of naturally producing laccases and white rot fungi are one such group In thepresent study three fungal species namely Ganoderma lucidum-CDBT1 Ganoderma japonicum and Lentinula edodes isolatedfrom their native habitat in Nepal were screened for laccase production andG lucidum-CDBT1 was found to express highest levelsof enzyme (day 10 culture media showed 092 IUmg total protein or 92 IUmL laccase activity with ABTS as substrate) Ligninextracted from rice straw was used in Olga medium for laccase production and isolation from G lucidum-CDBT1 Presence oflignin (5 gL) and copper sulfate (30 120583M) in the media increased the extracellular laccase content by 111 and 114 respectivelyThe laccase enzyme produced by G lucidum-CDBT1 was fractionated by ammonium sulfate and purified by DEAE Sepharoseanion exchange chromatography The purified enzyme was found to have a molecular mass of 43 kDa and exhibits optimal activityat pH 50 and 30∘C The isolated laccase was thermally stable for up to 70∘C for 1 h and exhibited broad pH stability The kineticconstants119870

119898119881max and119870cat determined using 221015840-azinobis-(-3-ethylbenzothiazoline-6-sulfonic acid) as substrate were found to

be 110120583M 36120583molminmg and 246minminus1 respectively The isolated thermostable laccase will be used in future experiments fordelignification process

1 Introduction

Laccases (benzenediol oxygen oxidoreductase EC 11032)are copper containing enzymes that catalyze one-electronoxidation of a wide variety of substrates such as diphenolspolyphenols diamines aromatic amines and other electronrich compounds using molecular O

2[1] They belong to the

family of proteins that include ascorbate oxidase ceruloplas-min and bilirubin oxidaseThese enzymes are believed to uti-lize a free radical-catalyzed reactionmechanism in which thesubstrate forms an unstable free radical that further under-goes nonenzymatic reactions including hydration anddispro-portionation reactions [2]

Laccases are distributed among some plants [3 4] fungi[3] and bacteria [5 6] Literature review shows that laccasesare widely distributed among the prokaryotes For exam-ple laccases from Azospirillum lipoferum [7] Marinomonasmediterranea [8] Streptomyces griseus [9] E coli [10] Bacillussubtilis [11] and many more bacteria have been purifiedand characterized The crystal structures for several bacteriallaccases have also been published [12ndash14] Laccases are rec-ognized for their wide spread applications including ethanolproduction food industry dye bleaching paper and pulpprocessing and production of value added chemicals fromlignin [12] Other applications of laccases include their usein detection of catecholamine neurotransmitters (dopamine

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 3238909 10 pageshttpdxdoiorg10115520163238909

2 BioMed Research International

(a) (b)

Figure 1 Ganoderma lucidum-CDBT1 fruiting bodies (courtesy of Central Department of Biotechnology) (a) ventral surface and (b) dorsalsurface

norepinephrine) and glucose dehydrogenase-coupled oxida-tion ofmorphine Some fungal laccases have also been shownto detoxify fungal metabolites for example aflatoxin B1 andaccordingly are useful in the field of food microbiology fromthe view point of food processing and preservation [15]

Among the fungi the wood-rotting basidiomycetes pro-duce different kinds of extracellular oxidoreductases includ-ing laccases peroxidases and oxidases that generate H

2O2

[16] The laccases from wood-rotting basidiomycetes are ofsignificant interest as they are able to utilize a wide spectrumof carbon sources including intermediates of lignin degra-dation phenols and heterocyclic compounds Like bacteriallaccases the fungal enzymes are also involved in efficientdegradation of lignin and various types of dyes such asazo heterocyclic reactive and polymeric dyes [17ndash19]Lignin is the most recalcitrant components in lignocellulosicbiomass that encases the cellulose and hemicellulose thusnecessitating pretreatment of lignocellulosic biomass to liber-ate fermentable sugars to produce liquid biofuels Given thelignin depolymerizing activity of laccases they can serve asefficient biocatalysts for the pretreatment of lignocellulosicbiomass to isolate fermentable sugars and other products [17ndash19]

Chemical pretreatment processes are available to releasethe fermentable sugars however they are expensive difficultto operate and environmentally unfriendly Further furanderivatives weak acids and phenolic compounds formedafter lignin breakdown during chemical delignification haveinhibitory effects on fermentation process [20 21] On theother hand biological methods provide far safer and eco-nomic alternatives to obtain fermentable sugars from ligno-cellulosic biomass One such alternative is depolymerizationof lignin with the aid of laccases There are numerous fungalspecies that grow on wooden logs and most of them secretelaccases to overcome the lignin barrier and thus obtain theenergy from cellulose and hemicelluloses [3 17ndash19] Themountainous regions ofNepal are a rich source of fungi grow-ing on logs [22] In this regard the Central Department ofBiotechnology at Tribhuvan University Kathmandu Nepalhas collected developed seed cultures and archived a numberof fungal species We believe that these fungal species haveadapted to their natural environment of Nepal and some of

themmay be efficient laccase producers Accordingly as partof this study we have screened three fungal species to identifypotent laccase producers optimized enzyme productionconditions and purified and characterized a laccase enzymefrom G lucidum-CDBT1

2 Materials and Methods

21Materials ABTS [221015840-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] and guaiacolwere purchased fromHi-mediaPvt Ltd New Delhi India DEAE Sepharose was purchasedfrom Sigma Chemical Co St Louis MO USA Proteinmolecular weight markers were purchased from Genei PvtLtd Bangalore India All other reagents and chemicals usedwere of analytical grade available locally

22 Fungal Species Cultures Pure cultures of Ganodermalucidum-CDBT1 (Figure 1) Ganoderma japonicum-CDBT2and Lentinula edodes-CDBT3 (locally known as Shiitake)preserved at 4∘C were obtained from Central Departmentof Biotechnology Tribhuvan University Nepal They weremaintained by subculturing them in potato dextrose agar(PDA) Petri-plates by incubating the cultures at 25∘C for 72 hin the case of Ganoderma species and 120 h for Lentinulaspecies Regarding the fungal species used herein althoughnot fully characterized we do believe that they are novelstrains adapted to growing in high altitudes of Nepal

23 Screening of Fungal Species for Laccase Production PDA-agar plates supplemented with guaiacol (002) (wtwt) 1-naphthol (5mM) and tannic acid (05) (wtwt) and inocu-lated with various fungal cultures were used for screening oflaccase production by the selected fungal species All cultureswere incubated at 25∘C Laccase secretion was monitored byvisual color change in the plates due to oxidation of screeningagents for several days [28 29] Biotic and abiotic cultureswere used as positive and native controls respectively Areddish brown color was formedwhen secreted laccases reactwith guaiacol a deep purple color was formed when theyreact with 1-naphthol and a brown color was formed whenlaccases react with tannic acid Guaiacol was added to the

BioMed Research International 3

media before autoclaving tannic acid was autoclaved sep-arately before addition to the media and 1-naphthol wasautoclaved along with the media

24 Lignin Isolation Lignin was isolated from rice straw asdescribed by Minu and associates [30] Dried rice straw waspowdered and oven dried overnight at 105∘C It was thenhydrolyzed at 120∘C for 60min with 1 (wtwt) sulfuric acidand the resulting residuewas then subjected to delignificationprocess at 120∘C for 60min with alkaline peroxide [15 (wtwt) NaOH and 05 (wtwt) H

2O2] In all hydrolysis steps

the total solids used were 10 (wtwt) and remaining 90included 89 (wtwt) water and 1 (wtwt) sulfuric acid Inthe delignification step the total solids used were 10 (wtwt)acid treated rice straw and remaining 90 was made up of88 (wtwt) water and 15 (wtwt) NaOH and 05 (wtwt)H2O2 The solubilized lignin also known as black liquor was

separated from the solids Lignin was isolated from the blackliquor by two-step treatments In the first step the pH wasdecreased to 70 and vacuum filtered to remove silica In thesecond step the liquid was further titrated with acid to dropthe pH to 30 and incubated overnight to precipitate ligninThe precipitated lignin was washed vacuum filtered anddried

25 Laccase Enzyme Assay Laccase enzyme activity wasmeasured at room temperature using ABTS as substrate(1mM solution prepared in 01M of sodium acetate bufferpH 50) A typical reaction mixture consisted of 350 120583L ofenzyme preparation (isolated or purified and appropriatelydiluted) and 350 120583L of 1mM ABTS and the final volume wasadjusted to 115mL with 01M of sodium acetate buffer pH50 Reaction was started by the addition of enzyme andoxidation of ABTS was monitored spectrophotometrically at420 nm for 90 seconds using quartz cuvettes The amount ofenzyme required to oxidize 1 120583mol ABTS per minute isconsidered equivalent to 1 unit of enzyme activity (120576420 =36000Mminus1 cmminus1 and path length l = 1 cm) [31]

26 Optimization of Laccase Production from Ganodermalucidum-CDBT1 Modified Olga medium [4 gL glucose3 gL peptone 06 gL K

2HPO4 04 gL KH

2PO4 1 mgL

ZnSO4 5mgL FeSO

4 and 05 gLMnSO

4 05mgLMgSO

4

05mgL CuSO4 and 5 gL lignin] was used for optimization

and production of laccase from G lucidum-CDBT1 [32]Four parameters namely (i) presence of lignin (controlexperiments contained no lignin in the media) (ii) tem-perature (range 20 to 50∘C varied at an interval of 10∘C)(iii) pH (varied between pH 30 and 80 at an interval of05 pH units) and (iv) copper sulfate concentration (variedbetween 10 120583M to 50120583M at an interval of 10 120583M controlexperiments contained no copper sulfate in the media) wereused to optimize laccase production The pH of the mediawas varied using 01M sodium acetate (pH 3ndash6) 01Msodium phosphate (pH 65ndash75) or 01M Tris-HCl (gtpH 75)Each experimental culture consisted of 50mL modified Olgamedia and 5 discs of actively growing G lucidum-CDBT1mycelium (5-day-old culture 7mm diameter discs) under

varied conditions (above) and they were incubated in anorbital shaker incubator at 160ndash200 rpm at room temperature(sim25∘C) except when the temperatures were varied Prelimi-nary experiments suggested a concentration of 5 gL ligninwas optimal for the production of laccase accordingly allcultures with the exception of the controls were grown in thepresence of 5 gL of lignin isolated from rice straw Laccaseenzyme activity in the culture media was monitored at 48-hour (2-day) intervals

27 Purification of Laccase Laccase secreted into the culturemedia was purified in three different steps First the cultureswere vacuum filtered using Whatman number 1 filter paperThus obtained supernatant was centrifuged at 10000timesg for10min to remove any debris and the clear supernatant wasfurther used for purification process [33] Second the super-natant was subjected to ammonium sulfate fractionation(40 to 80) The precipitated protein was isolated by cen-trifugation (10000timesg10min) The precipitates were recon-stituted in 01M sodium acetate buffer pH 50 and screenedfor the presence of laccase activity as described aboveThe fractions with high laccase activity were subjected todialysis overnight against 01M sodium phosphate buffer pH70 Finally the desalted enzyme factions were subjected to aDEAE Sepharose anion exchange column chromatography atpH 70 as described previously [25] The protein fractionscollected were analyzed for laccase activity and the fractionswith high laccase activity were pooled and subjected toSDS- and native-PAGE to assess its purity and determine itsmolecular mass

28 Electrophoresis Molecular mass of laccase was deter-mined by SDS-PAGEusing standardmolecularmassmarkers(Genei India Pvt Ltd) according to Laemmlirsquos method[34] Polyacrylamide gels (6 stacking gel and 10 sep-arating gel) loaded with denatured and reduced proteinpreparations were subjected to electrophoresis at 25mA for3 hours at room temperature Proteins were visualized onPAGE gels using EZ-Visi Blue protein staining solution [5]Native-PAGE was also as described above except the gelswere run at 4∘C protein samples were not denatured orreduced and SDS was omitted from the gels The native-PAGE gels were stained with 5mM ABTS solution and 1guaiacol solutions for 15min to visualize laccase bands on thegels

29 Protein Quantitation Protein content in various prepa-rations used in this study were determined by Bradfordmethod [35] using BSA as standard

210 Physicochemical Characterization of Laccase The pHand temperature optima for the purified laccase were deter-mined as follows Purified laccase enzyme activity wasdetermined by varying the pH 20ndash80 or the temperature(20ndash80∘C) of the reaction under standard assay conditionsdescribed above To investigate the pH stability the purifiedenzyme was preincubated in buffers of pH 20ndash80 at room


Table 1 Summary of purification of laccase from Ganoderma lucidum-CDBT1lowast

Enzyme Total activity(U)

Protein(120583gmL)

Specific activity(Umg) Fold-purification

Culture media-filtrate 9722 1057 092 10Ammonium sulfate faction (after overnight dialysis) 311574 359 8685 944DEAE-anion exchange chromatography 266204 133 20100 2180lowastPreparation of the culture media-filtrate containing laccase and its ammonium sulfate fractionation and DEAE-anion exchange chromatography to isolatelaccase were as described in Materials and Methods Laccase enzyme was monitored using 1mM ABTS as substrate at room temperature (sim25∘C) and proteinconcentrations in various factions were determined by Bradford assay described in Materials and Methods

temperature for up to 240min before their enzyme activi-ties were determined Similarly to investigate the thermalstability the purified enzyme (dialyzed against 01M sodiumacetate buffer pH 50) was incubated in water baths at varioustemperatures between 30 and 80∘C for up to 180min Analiquot of the enzyme was withdrawn every 30min andassayed for enzyme activity as above MichaelisndashMentenkinetics were used to determine 119870

119898and 119881max using ABTS as

the substrate (001 to 10mM) and the kinetic constants (119870119898

and 119881max) were derived from Lineweaver-Burk plots [28]

211 Data Analysis Microsoft Excel and Prism GraphPadV 500 computer programs were used for data analysis Allvalues reported are average of triplicate experiments

3 Results

31 Screening Fungal Species for Laccase Activities Threewhite rot fungal species namely G lucidum-CDBT1 Gjaponicum-CDBT2 and L edodes-CDBT3 (Shiitake) werescreened for the secretion of laccase using PDA platessupplemented with 1-naphthol tannic acid and guaiacol[28 29] The oxidative polymerization of guaiacol formingreddish brown zones in the medium oxidation of tannic acidto brown color [29] and oxidation of 1-naphthol to a deeppurple complex [28] are a visual confirmation for the pres-encesecretion of laccase enzyme In this test all speciestested that is G lucidum-CDBT1 G japonicum-CDBT2and L edodes-CDBT3 (Shiitake) gave visual colors describedabove indicating secretion of extracellular laccase into thePDA medium G lucidum-CDBT1 formed the largest zone ofcoloration (Figure 2) followed by L edodes-CDBT3 (Shiitake)(Figure 2) and G japanicum-CDBT2 (data not presented)Accordingly G lucidum-CDBT1 was chosen for the furtherstudy

32 Optimization of Laccase Production Media Effect ofLignin Copper Sulfate Temperature and pH Presence oflignin and copper sulfate is known to promote productionsecretion of laccase In this regard the culture media weresupplemented with 5 gL lignin (isolated from rice straw)This resulted in 111 increase in the secretion of laccase as

compared to a control culture supplemented with glucose(Figure 3) The effect of copper sulfate on laccase pro-duction was determined by varying its concentration (10ndash50 120583M) in the culturemediumMaximum laccase production(93 IUmL on 10th day a 114 increase as compared to theblank) was obtained at a concentration of 30 120583M copper sul-fate (Figure 3)G lucidum-CDBT1 cultures were further sub-jected to temperature and pH optimization studies to deter-mine the conditions required for the optimal secretion oflaccase The optimal temperature and pH needed to producehigh levels of laccase were found to be 30∘C (89 IUmL on day10) and pH 50 (92 IUmL on day 10) respectively (Figure 3)Secretion of detectable levels of the enzyme activity beginsas early as day 2 and reaches a maximum at around day 10Enzyme activity decreased sharply as the pH increased from50 towards the neutral range or the temperature increasedtowards 50∘C (Figure 3)

33 Purification of Laccase Theculture filtrate obtained fromculture media subjected to ammonium sulfate precipitationled to enrichment of laccase in the 70 ammonium sulfatefaction The precipitate dissolved in sodium acetate bufferpH 50 and dialyzed overnight was further purified byDEAE Sepharose anion exchange column chromatographyTable 1 The column was developed using a linear gradientof 0 to 01M sodium chloride at pH 70 The peak fractionswere pooled and analyzed by native- and denaturing-PAGE(Figure 4) The ion-exchange chromatography purified lac-case protein pool showed the presence of a single proteinband on SDS-PAGE gels with a molecular weight of 43 kDa(Figure 4(a)) Native-PAGE gels stained with ABTS andguaiacol as substrates also indicated the presence of oneisozyme of laccase (Figures 4(b) and 4(c)) in the purifiedfaction The extracts as judged by native-PAGE showedthe presence of 3 laccase isozymes and the purified enzymecorresponds to isozyme 3 (the major isoform produced)

34 Partial Characterization of Purified Laccase pH and Tem-perature Optima and pH and Thermal Stability The influ-ence of pH and temperature on laccase activity was deter-mined by varying pH of the reaction mixture from pH 20to 80 while the influence of temperature on laccase activitywas determined by varying the temperature of the reactionbetween 20 and 80∘C and at pH 50 (pH optima) The pH


Ganoderma lucidum-CDBT1 Lentinula edodes-CDBT3 (Shiitake)

Subs

trate

1-n

apht

hol

Subs

trat

e ta

nnic

acid

Subs

trat

e gu

aiac

ol

(a)

(b)

(c)

Figure 2 Screening of Ganoderma lucidum-CDBT1 and Lentinula edodes-CDBT3 (Shiitake) for secretion of laccase The experimentalconditions were as described in Materials and Methods and stained with 1-naphthol (a) tannic acid (b) and guaiacol (c)

and temperature optima for laccases were found to be pH50 and 30∘C respectively (Figures 5(a) and 5(b)) Laccasewas active in a wide range of pH values At pH values abovepH 50 the enzyme activity decreased gradually by 50 atpH 8 (Figure 5(a)) Laccase activity was relatively stable inthe range of pH 30ndash70 The enzyme activity declined whenthe temperature was increased from 30 to 80∘C (Figure 5(b))Purified laccase was incubated in buffers of varying pHbetween pH 20 and 80 for up to 180min to determine itspH stability Similarly the purified enzyme dissolved in 01Msodium acetate pH 50 was incubated at varying tempera-tures (40ndash80∘C) for up to 180min to determine its thermalstability Laccase was found to be most stable at pH 50and 30∘C (Figures 5(c) and 5(d)) Laccase activity decreasedsignificantly (93) after 180min at pH 80 and it wasfairly stable between pH 30 and 70 for up to 180minWhile the enzyme was relatively stable below 50∘C activity

decreased significantly when the temperature was 70∘C orhigher In fact laccase activity was completely lost within anhour at 80∘C (Figures 5(c) and 5(d))

35 Determination of Kinetic Parameters The kinetic param-eters (119870

119898 119881max and 119870cat) for the purified laccase enzyme

were determined using varied concentrations ofABTS as sub-strate and they were found to be 110 120583M 36 120583molminmgand 246minminus1 respectively

4 Discussion

Someof themost important factors that influence secretion oflaccase into culture media by Ganoderma species include (i)pH and temperature for their growth (ii) addition of suitableamounts of sugars for example glucose (carbon source)


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Figure 3 Optimization of experimental conditions for laccase production in Ganoderma lucidum-CDBT1 Modified Olga medium was usedfor G lucidum-CDBT1 under various conditions shown in (a)ndash(d) as described in Materials and Methods Experiments described in (a) (b)and (c) were performed at room temperature (sim25∘C)ThemodifiedOlgamediumused to growG lucidum (experiments described in (a) (c)and (d)) contained 05 gL CuSO

4and 5 gL lignin (experiments described in (b) (c) and (d)) (a) Effect of 5 gL lignin on laccases secretion

(b) effect of CuSO4on laccase secretion (c) effect of pH of the media on secretion of laccase and (d) effect of incubation temperature on

the secretion of laccases Enzyme activity was determined as described in Materials and Methods With the exception of the experimentsinvolving the effect of media pH on secretion of laccase all other experiments were performed at pH 50

(iii) addition of lignin (inducer) and (iv) an appropriateconcentration of copper sulfate (inducer) The optimal pHconditions under which fungal laccases are secreted into cul-turemedia vary between pH 30 and 70This also depends onthe type of substrate used in the activity assays For examplewhen ABTS is used as substrate the pH optima are more inacidic range pH 30ndash50 [1] In our study laccase secretionwas found to be maximal at pH 50 Accordingly all of thecultures used for the production of laccase from G lucidum-CDBT1 in this study were performed at pH 50 Further theenzymewas secreted into the culturemedia over a wide rangeof pH suggesting versatility of the enzyme for applications in

many biotechnological processes The decrease in activity athigher pH is most probably due to the binding of a hydroxideanion to type 2 and 3 copper centers in laccase which in turninhibits the binding of oxygen to the copper centers and thusdecreased laccase activity [35 36]

G lucidum-CDBT1 cultures used in this study secretedmaximal laccase into the culture medium at 30∘C This hasbeen supported by the fact that G lucidum is a mesophilicfungus and others have shown that G lucidum secretesmaximal laccase at 25∘C [25]

Addition of glucose to culture media has been shown toinfluence laccase synthesis [37]The concentration of glucose


66

43

29

14

(kDa)

1 2 1 2 1 2

(a) (b) (c)

Isozyme 1Isozyme 2

Isozyme 3

Figure 4 PAGE analysis of laccase isolated from Ganodermalucidum-CDBT1 (a) SDS-PAGE analysis of purified laccase (Lane 1marker proteins Lane 2 purified laccase protein) (b) and (c) native-PAGE analysis of laccase purified (Lane 1) or isolated from culturemedium (Lane 2) (b)The gel was stained with guaiacol as substrateand (c) the gel was stained with ABTS as substrate

used in these studies varied from 01 to 1 [38] The Glucidum-CDBT1 cultures used herein were optimized to growin relatively low concentrations (04) of glucose contain-ing media and under these conditions G lucidum-CDBT1culture produced relatively high levels of laccase while highconcentrations of sugars may satisfy the nutrient demands ofG lucidum for growth but do not necessarily promote optimalsecretion of laccase In certain species for example Trametespubescens laccase production was in fact inhibited by highconcentrations of glucose [39] Additionally it has also beenshown that high concentrations of glucose trigger synthesisof extracellular polysaccharides that will interfere with theextraction of laccase from culture media [40] Given theabove factors 04 glucose was used while optimizing ourcultures

Addition of lignin to culture media has also been shownto enhance laccase secretion [41] Our cultures were opti-mized to grow and secrete maximal amount of laccase intoculture media at concentrations of 5 gL lignin (05) Suchconcentrations resulted in two-fold increase in laccase activ-ity in cultures as compared to the culture media containingonly 04 glucose The latter may be due to the synthesis ofinducible laccase isozymes which in turn is believed to bebecause of increased secondary metabolism Sharma andassociates [41] have also observed an increase in laccase yieldin aGanoderma sp rckk-02 when cultured in the presence ofincreasing concentrations of ligninOther compounds shownto increase laccase production in cultures include veratrylalcohol syringic acid and 25-xylidine [41ndash43] We usedpeptone as a nitrogen source which is known to enhancelaccase synthesis [38] Due to the complex composition ofpeptones they provide a wide range of benefits to the cellsand cell performance Copper ions (Cu2+) are part of theactive site of laccases accordingly CuSO

4is most frequently

supplemented with growthmedia to enhance laccase produc-tion in fungi Cu2+ ions are thus crucial for the synthesis of

catalytically active laccase protein Further Cu2+ ions alsoinduce the expression of certain genes including laccase gene[44] The promoter region of the genes encoding for laccasecontains various recognition sites specific for xenobioticsand heavy metals [3 45] It has been demonstrated that thePleurotus laccase genes poxc and poxa1b are transcriptionallyinduced by Cu2+ ions [45 46] Relatively high concentrationsof Cu2+ have also been shown to suppress laccase productionwhich could be due to activation of defense mechanisms[42] In our experiments the optimal concentration of Cu2+required to express highest levels of laccases was 30 120583MThis is consistent with other studies [45 46] In addition toCu2+ distinct organic inducers with structural similaritiesor relationships with lignin are often used to induce theexpression of laccase [47]

Many fungal laccase isozymes have been purified andtheir molecular properties have been summarized [3] Thelaccase enzyme purified from G lucidum-CDBT1 had amolecular mass of 43 kDa and exhibited 119870

119898 119881max and

119870cat values of 0110mM 36 120583molminmg and 246minminus1respectively The 119870

119898values for laccases reported previously

ranged from 01 to 37mM with ABTS as substrate [23] The119870119898and 119881max values of recombinant laccase heterologously

expressed in Pichia pastoris using ABTS substrate were foundto be 0521mM and 1965 120583molminmg respectively [3 2348] 119870

119898reported in this work is lower than most other

reported values suggesting this enzyme has higher affinitytowards nonphenolic substrate ABTS [47]This is as expectedas these enzymes originate from different organisms andtheir physicochemical characteristics have been shown to bedifferent The other physicochemical characteristics forexample pH and temperature optima of the laccase isozymeisolated from G lucidum-CDBT1 were similar but not nec-essarily identical to those previously reported [25 47] Theisolated enzyme was thermally stable (70∘C for up to 1 h) andexhibited broad pH stability (pH 30ndash70)

Fungal species express a number of different laccaseisozymes with molecular masses ranging from 24 to 80 kDa[40] and this variation could be attributed to the differentecological origins of the species or different culture condi-tions under which various isozymes were expressed andorsecreted In particular presence of the inducers in the mediaseems to play an important role in which laccase isozymeswere secreted into the medium For example P pulmonariusproduced three laccase isozymes two of which (lcc1 and lcc2)were constitutive and the 3rd is inducible (lcc3) lcc3 wasdetected only when the fungus was cultured in the presenceof inducers [48] This also appears to be true among variousstrains of G lucidum Table 2 In this study native-PAGEanalysis suggested the presence of three laccase isozymes inG lucidum-CDBT1 As judged by native-PAGE two of theisozymes were minor components whereas the 3rd was amajor inducible isozyme The major isozyme has been suc-cessfully purified in this study This enzyme has character-istics similar to the enzyme isolated by Ko and associates[25] although the molecular mass is different Molecularweight of the laccase isozyme-3 (Figure 4) reported herein(43 kDa as judged by SDS-PAGE) is in agreement with the


Table 2 A comparison of the physicochemical characteristics of laccases isolated from several strains of Ganoderma lucidum

G lucidum strain Mol mass (kDa) pH optimum 119870119898with ABTS as substrate (mM) Country of origin Reference

Unknown 68 30 0114 China [23]CBS22993 625 50 0107 Netherlands [24]Galc3 65ndash68 35 0037 South Korea [25]IBL-05 383 50 0047 India [26]MDU-7 24ndash66 50 0026ndash0029 India [27]CDBT1 43 50 0110 Nepal This work

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(d) Temperature stability

Figure 5 pH and temperature optimum and pH and thermal stability of purified laccase The experimental conditions were as described inMaterials andMethods Experiments described in (a) and (c) were performed at room temperature (sim25∘C)The experimental results depictedin (a) and (c) (pH optimum and pH stability) were performed using buffers of various pH specified whereas the temperature optimum andthermal stability was performed at pH 5 (01M sodium acetate pH 50)


value reported by Murugesan and associates [49] based onSDS- and native-PAGE of crude extracts stained for enzymeactivity This study [49] did not undertake physicochemicalcharacterization of the enzyme The minor isoforms that wehave observed in native-PAGE gels are being isolated usingenrichment processes in an ongoing study Ko and associates[25] have in fact reported the presence of three laccaseisozymes in G lucidum whose molecular masses rangedbetween 40 and 68 kDa [42]The number of laccase isozymessecreted by other fungal species also varies For example Tmulticolor is reported to secrete five isozymes [50] whereas Postreatus secretes eight different laccase isozymes in responseto Cu2+ ions [51]The number of laccase isozymes expressedproduced by different strains of the same species was also dif-ferent [23ndash27 37] In this regard we have summarized somephysicochemical characteristics of several laccases iso-lated from a number of different strains of G lucidumTable 2 Accordingly it appears that the laccase isozyme 3is isolated and purified in this study although having similarenzyme characteristics its molecular mass is different Fur-thermolecular characterization for example sequence deter-mination and its comparison to other know sequences isrequired to call this a novel enzyme

In conclusion we have successfully cultured and opti-mized culture conditions induced expression and purifiedand characterized a laccase in G lucidum-CDBT1 isolatedfrom its native habitat in Nepal The isozyme characterizedfrom G lucidum-CDBT1 appears to be different from thosereported among various strains of G lucidum Identificationof minor isoforms of laccases inG lucidum-CDBT1 and vari-ous laccases present in other organisms screened in this studythat is G japonicum and L edodes and the use of theseenzymes to depolymerize lignin to expose cellulose andhemicellulose fibers from lignocellulosic biomass for efficienthydrolysis and fermentation of sugars are being studied in ourlaboratory

Competing Interests

The authors hereby declare that they have no commercial orother competing andor conflicting interests with regard tothe publication of this research paper

References

[1] V Madhavi and S S Lele ldquoLaccase properties and applica-tionsrdquo BioResources vol 4 no 4 pp 1694ndash1717 2009

[2] H Claus ldquoLaccases structure reactions distributionrdquoMicronvol 35 no 1-2 pp 93ndash96 2004

[3] P Baldrian ldquoFungal laccases-occurrence and propertiesrdquo FEMSMicrobiology Reviews vol 30 no 2 pp 215ndash242 2006

[4] R S Shekhar S Sehgal M Kamthania and A Kumar ldquoLac-case microbial sources production purification and potentialbiotechnological applicationsrdquoEnzyme Research vol 2011 Arti-cle ID 217861 11 pages 2011

[5] K Hilden T K Hakala P Maijala T K Lundell and AHatakka ldquoNovel thermotolerant laccases produced by thewhite-rot fungus Physisporinus rivulosusrdquo Applied Microbiologyand Biotechnology vol 77 no 2 pp 301ndash309 2007

[6] P Sharma R Goel and N Capalash ldquoBacterial laccasesrdquoWorldJournal of Microbiology and Biotechnology vol 23 no 6 pp823ndash832 2007

[7] G Diamantidis A Effosse P Potier and R Bally ldquoPurificationand characterization of the first bacterial laccase in the rhi-zospheric bacterium Azospirillum lipoferumrdquo Soil Biology andBiochemistry vol 32 no 7 pp 919ndash927 2000

[8] F Solano E Garcıa D Perez and A Sanchez-Amat ldquoIsolationand characterization of strain MMB-1 (CECT 4803) a novelmelanogenic marine bacteriumrdquo Applied and EnvironmentalMicrobiology vol 63 no 9 pp 3499ndash3506 1997

[9] K Endo Y Hayashi T Hibi K Hosono T Beppu and KUeda ldquoEnzymological characterization of EpoA a laccase-likephenol oxidase produced by Streptomyces griseusrdquo Journal ofBiochemistry vol 133 no 5 pp 671ndash677 2003

[10] CKimWW Lorenz J THoopes and J FDDean ldquoOxidationof phenolate siderophores by the multicopper oxidase encodedby the Escherichia coli yacK generdquo Journal of Bacteriology vol183 no 16 pp 4866ndash4875 2001

[11] N P Muthukumarasamy B Jackson A Joseph Raj and MSevanan ldquoProduction of extracellular laccase from Bacillussubtilis MTCC 2414 using agroresidues as a potential sub-straterdquoBiochemistry Research International vol 2015 Article ID765190 9 pages 2015

[12] P Giardina V Faraco C Pezzella A Piscitelli S Vanhulleand G Sannia ldquoLaccases a never-ending storyrdquo Cellular andMolecular Life Sciences vol 67 no 3 pp 369ndash385 2010

[13] M Gunne A Hoppner P-L Hagedoorn and V B UrlacherldquoStructural and redox properties of the small laccase Ssl1 fromStreptomyces sviceusrdquo The FEBS Journal vol 281 no 18 pp4307ndash4318 2014

[14] SMajumdar T Lukk J O Solbiati et al ldquoRoles of small laccasesfrom Streptomyces in lignin degradationrdquo Biochemistry vol 53no 24 pp 4047ndash4058 2014

[15] J F Alberts W C A Gelderblom A Botha and W H vanZyl ldquoDegradation of aflatoxin B1 by fungal laccase enzymesrdquoInternational Journal of Food Microbiology vol 135 no 1 pp47ndash52 2009

[16] D Wesenberg I Kyriakides and S N Agathos ldquoWhite-rotfungi and their enzymes for the treatment of industrial dyeeffluentsrdquo Biotechnology Advances vol 22 no 1-2 pp 161ndash1872003

[17] K Murugesan I-H Yang Y-M Kim J-R Jeon and Y-S Chang ldquoEnhanced transformation of malachite green bylaccase of Ganoderma lucidum in the presence of naturalphenolic compoundsrdquo Applied Microbiology and Biotechnologyvol 82 no 2 pp 341ndash350 2009

[18] T K Kirk andR L Farrell ldquoEnzymatic lsquocombustionrsquo themicro-bial degradation of ligninrdquo Annual Review of Microbiology vol41 pp 465ndash501 1987

[19] L Gianfreda F Xu and J-M Bollag ldquoLaccases a useful groupof oxidoreductive enzymesrdquo Bioremediation Journal vol 3 no1 pp 1ndash26 1999

[20] B Joshi M R Bhatt D Sharma J Joshi R Malla and LSreerama ldquoLignocellulosic ethanol production current prac-tices and recent developmentsrdquo Biotechnology and MolecularBiology Reviews vol 6 no 8 pp 172ndash182 2011

[21] J R M Almeida T Modig A Petersson B Hahn-HagerdalG Liden and M-F Gorwa-Grauslund ldquoIncreased toleranceand conversion of inhibitors in lignocellulosic hydrolysates bySaccharomyces cerevisiaerdquo Journal of Chemical Technology andBiotechnology vol 82 no 4 pp 340ndash349 2007


[22] M Christensen S Bhattarai S Devkota and H O LarsenldquoCollection and use of wild edible fungi in Nepalrdquo EconomicBotany vol 62 no 1 pp 12ndash23 2008

[23] Z Ding L Peng Y Chen et al ldquoProduction and characteriza-tion of thermostable laccase from the mushroom Ganodermalucidum using submerged fermentationrdquo African Journal ofMicrobiology Research vol 6 no 6 pp 1147ndash1157 2012

[24] T Manavalan A Manavalan K P Thangavelu and K HeeseldquoCharacterization of optimized production purification andapplication of laccase from Ganoderma lucidumrdquo BiochemicalEngineering Journal vol 70 no 1 pp 106ndash114 2013

[25] E-M Ko Y-E Leem and H T Choi ldquoPurification and charac-terization of laccase isozymes from the white-rot basidiomyceteGanoderma lucidumrdquo Applied Microbiology and Biotechnologyvol 57 no 1-2 pp 98ndash102 2001

[26] M Bilal and M Asgher ldquoEnhanced catalytic potentiality ofGanoderma lucidum IBL-05 manganese peroxidase immobi-lized on sol-gel matrixrdquo Journal of Molecular Catalysis BEnzymatic vol 128 no 6 pp 82ndash93 2016

[27] A Kumar K K Sharma P Kumar and N Ramchiary ldquoLaccaseisozymes from Ganoderma lucidum MDU-7 isolation char-acterization catalytic properties and differential role duringoxidative stressrdquo Journal of Molecular Catalysis B Enzymaticvol 113 no 3 pp 68ndash75 2015

[28] S S More P S Renuka K Pruthvi M Swetha S Malini andS M Veena ldquoIsolation purification and characterization offungal laccase from Pleurotus sprdquo Enzyme Research vol 2011Article ID 248735 7 pages 2011

[29] L-L Kiiskinen M Ratto and K Kruus ldquoScreening for novellaccase-producing microbesrdquo Journal of Applied Microbiologyvol 97 no 3 pp 640ndash646 2004

[30] K Minu K K Jiby and V V N Kishore ldquoIsolation andpurification of lignin and silica from the black liquor generatedduring the production of bioethanol from rice strawrdquo Biomassand Bioenergy vol 39 no 4 pp 210ndash217 2012

[31] B SWolfenden and R LWillson ldquoRadical-cations as referencechromogens in kinetic studies of one-electron transfer reac-tionsrdquo Journal Chemical Society Perkin Transactions vol 2 pp805ndash812 1982

[32] O V Koroljova-Skorobogatrsquoko E V Stepanova V P Gavrilovaet al ldquoPurification and characterization of the constitutive formof laccase from the basidiomycete Coriolus hirsutus and effectof inducers on laccase synthesisrdquo Biotechnology and AppliedBiochemistry vol 28 no 1 pp 47ndash54 1998

[33] M-J Han H-T Choi and H-G Song ldquoPurification andcharacterization of laccase from the white rot fungus Trametesversicolorrdquo Journal of Microbiology vol 43 no 6 pp 555ndash5602005

[34] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[35] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein-dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[36] F Xu ldquoEffects of redox potential and hydroxide inhibition onthe pH activity profile of fungal laccasesrdquo Journal of BiologicalChemistry vol 272 no 2 pp 924ndash928 1997

[37] C Teerapatsakul N Abe C Bucke N Kongkathip S Jareonkit-mongkol and L Chitradon ldquoNovel laccases of Ganoderma sp

KU-Alk4 regulated by different glucose concentration in alka-line mediardquo World Journal of Microbiology and Biotechnologyvol 23 no 11 pp 1559ndash1567 2007

[38] V V Kumar S D Kirupha P Periyaraman and S SivanesanldquoScreening and induction of laccase activity in fungal speciesand its application in dye decolorizationrdquo African Journal ofMicrobiology Research vol 5 no 11 pp 1261ndash1267 2011

[39] C Galhaup H Wagner B Hinterstoisser and D HaltrichldquoIncreased production of laccase by the wood-degrading basid-iomycete Trametes pubescensrdquo Enzyme and Microbial Technol-ogy vol 30 no 4 pp 529ndash536 2002

[40] C Eggert U Temp and K-E L Eriksson ldquoThe ligninolyticsystem of the white rot fungus Pycnoporus cinnabarinuspurification and characterization of the laccaserdquo Applied andEnvironmental Microbiology vol 62 no 4 pp 1151ndash1158 1996

[41] K K Sharma B Shrivastava V R B Sastry N Sehgal and RC Kuhad ldquoMiddle-redox potential laccase from Ganodermasp its application in improvement of feed for monogastricanimalsrdquo Scientific Reports vol 3 article 1299 2013

[42] K Rajendran M S M Annuar and M A A Karim ldquoMediumoptimization for fungal laccase productionrdquoAsia Pacific Journalof Molecular Biology and Biotechnology vol 19 no 2 pp 73ndash812011

[43] T M DrsquoSouza C S Merritt and C A Reddy ldquoLignin-modifying enzymes of the white rot basidiomycete Ganodermalucidumrdquo Applied and Environmental Microbiology vol 65 no12 pp 5307ndash5313 1999

[44] J-W ParkH-WKang B-S Ha S-I Kim S Kim andH-S RoldquoStrain-dependent response to Cu2+ in the expression of laccasein Pycnoporus coccineusrdquo Archives of Microbiology vol 197 no4 pp 589ndash596 2015

[45] P Baldrian ldquoInteractions of heavy metals with white-rot fungirdquoEnzyme andMicrobial Technology vol 32 no 1 pp 78ndash91 2003

[46] G Palmieri P Giardina C Bianco B Fontanella andG SannialdquoCopper induction of laccase isoenzymes in the ligninolyticfungus Pleurotus ostreatusrdquo Applied and Environmental Micro-biology vol 66 no 3 pp 920ndash924 2000

[47] S R Couto M Gundın M Lorenzo and M A SanromanldquoScreening of supports and inducers for laccase productionby Trametes versicolor in semi-solid-state conditionsrdquo ProcessBiochemistry vol 38 no 2 pp 249ndash255 2002

[48] L-F You Z-M Liu J-F Lin L-Q Guo X-L Huang and H-X Yang ldquoMolecular cloning of a laccase gene from Ganodermalucidum and heterologous expression in Pichia pastorisrdquo Journalof Basic Microbiology vol 54 no 1 pp S134ndashS141 2014

[49] K Murugesan I-H Nam Y-M Kim and Y-S Chang ldquoDecol-orization of reactive dyes by a thermostable laccase producedby Ganoderma lucidum in solid state culturerdquo Enzyme andMicrobial Technology vol 40 no 7 pp 1662ndash1672 2007

[50] C Leitner J Hess C Galhaup et al ldquoPurification and char-acterization of a laccase from the white-rot fungus Trametesmulticolorrdquo Applied Biochemistry and BiotechnologymdashPart AEnzyme Engineering and Biotechnology vol 98ndash100 pp 497ndash507 2002

[51] O V Morozova G P Shumakovich M A Gorbacheva S VShleev and A I Yaropolov ldquolsquoBluersquo laccasesrdquo Biochemistry vol72 no 10 pp 1136ndash1150 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


(a) (b)

Figure 1 Ganoderma lucidum-CDBT1 fruiting bodies (courtesy of Central Department of Biotechnology) (a) ventral surface and (b) dorsalsurface

norepinephrine) and glucose dehydrogenase-coupled oxida-tion ofmorphine Some fungal laccases have also been shownto detoxify fungal metabolites for example aflatoxin B1 andaccordingly are useful in the field of food microbiology fromthe view point of food processing and preservation [15]

Among the fungi the wood-rotting basidiomycetes pro-duce different kinds of extracellular oxidoreductases includ-ing laccases peroxidases and oxidases that generate H

2O2

[16] The laccases from wood-rotting basidiomycetes are ofsignificant interest as they are able to utilize a wide spectrumof carbon sources including intermediates of lignin degra-dation phenols and heterocyclic compounds Like bacteriallaccases the fungal enzymes are also involved in efficientdegradation of lignin and various types of dyes such asazo heterocyclic reactive and polymeric dyes [17ndash19]Lignin is the most recalcitrant components in lignocellulosicbiomass that encases the cellulose and hemicellulose thusnecessitating pretreatment of lignocellulosic biomass to liber-ate fermentable sugars to produce liquid biofuels Given thelignin depolymerizing activity of laccases they can serve asefficient biocatalysts for the pretreatment of lignocellulosicbiomass to isolate fermentable sugars and other products [17ndash19]

Chemical pretreatment processes are available to releasethe fermentable sugars however they are expensive difficultto operate and environmentally unfriendly Further furanderivatives weak acids and phenolic compounds formedafter lignin breakdown during chemical delignification haveinhibitory effects on fermentation process [20 21] On theother hand biological methods provide far safer and eco-nomic alternatives to obtain fermentable sugars from ligno-cellulosic biomass One such alternative is depolymerizationof lignin with the aid of laccases There are numerous fungalspecies that grow on wooden logs and most of them secretelaccases to overcome the lignin barrier and thus obtain theenergy from cellulose and hemicelluloses [3 17ndash19] Themountainous regions ofNepal are a rich source of fungi grow-ing on logs [22] In this regard the Central Department ofBiotechnology at Tribhuvan University Kathmandu Nepalhas collected developed seed cultures and archived a numberof fungal species We believe that these fungal species haveadapted to their natural environment of Nepal and some of

themmay be efficient laccase producers Accordingly as partof this study we have screened three fungal species to identifypotent laccase producers optimized enzyme productionconditions and purified and characterized a laccase enzymefrom G lucidum-CDBT1

2 Materials and Methods

21Materials ABTS [221015840-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] and guaiacolwere purchased fromHi-mediaPvt Ltd New Delhi India DEAE Sepharose was purchasedfrom Sigma Chemical Co St Louis MO USA Proteinmolecular weight markers were purchased from Genei PvtLtd Bangalore India All other reagents and chemicals usedwere of analytical grade available locally

22 Fungal Species Cultures Pure cultures of Ganodermalucidum-CDBT1 (Figure 1) Ganoderma japonicum-CDBT2and Lentinula edodes-CDBT3 (locally known as Shiitake)preserved at 4∘C were obtained from Central Departmentof Biotechnology Tribhuvan University Nepal They weremaintained by subculturing them in potato dextrose agar(PDA) Petri-plates by incubating the cultures at 25∘C for 72 hin the case of Ganoderma species and 120 h for Lentinulaspecies Regarding the fungal species used herein althoughnot fully characterized we do believe that they are novelstrains adapted to growing in high altitudes of Nepal

23 Screening of Fungal Species for Laccase Production PDA-agar plates supplemented with guaiacol (002) (wtwt) 1-naphthol (5mM) and tannic acid (05) (wtwt) and inocu-lated with various fungal cultures were used for screening oflaccase production by the selected fungal species All cultureswere incubated at 25∘C Laccase secretion was monitored byvisual color change in the plates due to oxidation of screeningagents for several days [28 29] Biotic and abiotic cultureswere used as positive and native controls respectively Areddish brown color was formedwhen secreted laccases reactwith guaiacol a deep purple color was formed when theyreact with 1-naphthol and a brown color was formed whenlaccases react with tannic acid Guaiacol was added to the









2HPO4 04 gL KH

2PO4 1 mgL

ZnSO4 5mgL FeSO

4 and 05 gLMnSO

4 05mgLMgSO

4











Protein(120583gmL)








3 Results








Subs

trate

1-n

apht

hol

Subs

trat

e ta

nnic

acid

Subs

trat

e gu

aiac

ol

(a)

(b)

(c)







4 Discussion



0

25

50

75

100

0 3 6 9 12

Enzy

me a

ctiv

ity (I

Um

L)

Time (days)

No lignin5gL lignin


0

25

50

75

100

0 3 6 9 12

Enzy

me a

ctiv

ity (I

Um

L)

Time (days)

0 120583M10 120583M20 120583M

30 120583M40 120583M50 120583M


0

25

50

75

100

0 3 6 9 12

Enzy

me a

ctiv

ity (I

Um

L)

Time (days)

pH 3pH 4pH 5

pH 6pH 7pH 8


0

25

50

75

100

0 5 10 15

Enzy

me a

ctiv

ity (I

Um

L)

Time (days)

20∘C30∘C

40∘C50∘C











66

43

29

14

(kDa)

1 2 1 2 1 2

(a) (b) (c)

Isozyme 1Isozyme 2

Isozyme 3




4is most frequently




119898 119881max and












1000

1500

2000

2500

3000

3500

0 5 10

Enzy

me a

ctiv

ity (I

Um

L)

pH

(a) pH optimum

2000

2500

3000

3500

4000

4500

0 20 40 60 80 100

Enzy

me a

ctiv

ity (I

Um

L)

Temperature (∘C)


0

1000

2000

3000

4000

0 50 100 150 200

Enzy

me a

ctiv

ity (I

Um

L)

Time (min)

pH 2pH 3pH 4pH 5

pH 6pH 7pH 8

(c) pH stability

0

1000

2000

3000

0 50 100 150 200

Enzy

me a

ctiv

ity (I

Um

L)

Time (min)

40∘C50∘C60∘C

70∘C80∘C






Competing Interests


References





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









2HPO4 04 gL KH

2PO4 1 mgL

ZnSO4 5mgL FeSO

4 and 05 gLMnSO

4 05mgLMgSO

4











Protein(120583gmL)








3 Results








Subs

trate

1-n

apht

hol

Subs

trat

e ta

nnic

acid

Subs

trat

e gu

aiac

ol

(a)

(b)

(c)







4 Discussion



0

25

50

75

100

0 3 6 9 12

Enzy

me a

ctiv

ity (I

Um

L)

Time (days)

No lignin5gL lignin


0

25

50

75

100

0 3 6 9 12

Enzy

me a

ctiv

ity (I

Um

L)

Time (days)

0 120583M10 120583M20 120583M

30 120583M40 120583M50 120583M


0

25

50

75

100

0 3 6 9 12

Enzy

me a

ctiv

ity (I

Um

L)

Time (days)

pH 3pH 4pH 5

pH 6pH 7pH 8


0

25

50

75

100

0 5 10 15

Enzy

me a

ctiv

ity (I

Um

L)

Time (days)

20∘C30∘C

40∘C50∘C











66

43

29

14

(kDa)

1 2 1 2 1 2

(a) (b) (c)

Isozyme 1Isozyme 2

Isozyme 3




4is most frequently




119898 119881max and












1000

1500

2000

2500

3000

3500

0 5 10

Enzy

me a

ctiv

ity (I

Um

L)

pH

(a) pH optimum

2000

2500

3000

3500

4000

4500

0 20 40 60 80 100

Enzy

me a

ctiv

ity (I

Um

L)

Temperature (∘C)


0

1000

2000

3000

4000

0 50 100 150 200

Enzy

me a

ctiv

ity (I

Um

L)

Time (min)

pH 2pH 3pH 4pH 5

pH 6pH 7pH 8

(c) pH stability

0

1000

2000

3000

0 50 100 150 200

Enzy

me a

ctiv

ity (I

Um

L)

Time (min)

40∘C50∘C60∘C

70∘C80∘C






Competing Interests


References





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Protein(120583gmL)








3 Results








Subs

trate

1-n

apht

hol

Subs

trat

e ta

nnic

acid

Subs

trat

e gu

aiac

ol

(a)

(b)

(c)







4 Discussion



0

25

50

75

100

0 3 6 9 12

Enzy

me a

ctiv

ity (I

Um

L)

Time (days)

No lignin5gL lignin


0

25

50

75

100

0 3 6 9 12

Enzy

me a

ctiv

ity (I

Um

L)

Time (days)

0 120583M10 120583M20 120583M

30 120583M40 120583M50 120583M


0

25

50

75

100

0 3 6 9 12

Enzy

me a

ctiv

ity (I

Um

L)

Time (days)

pH 3pH 4pH 5

pH 6pH 7pH 8


0

25

50

75

100

0 5 10 15

Enzy

me a

ctiv

ity (I

Um

L)

Time (days)

20∘C30∘C

40∘C50∘C











66

43

29

14

(kDa)

1 2 1 2 1 2

(a) (b) (c)

Isozyme 1Isozyme 2

Isozyme 3




4is most frequently




119898 119881max and












1000

1500

2000

2500

3000

3500

0 5 10

Enzy

me a

ctiv

ity (I

Um

L)

pH

(a) pH optimum

2000

2500

3000

3500

4000

4500

0 20 40 60 80 100

Enzy

me a

ctiv

ity (I

Um

L)

Temperature (∘C)


0

1000

2000

3000

4000

0 50 100 150 200

Enzy

me a

ctiv

ity (I

Um

L)

Time (min)

pH 2pH 3pH 4pH 5

pH 6pH 7pH 8

(c) pH stability

0

1000

2000

3000

0 50 100 150 200

Enzy

me a

ctiv

ity (I

Um

L)

Time (min)

40∘C50∘C60∘C

70∘C80∘C






Competing Interests


References





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Subs

trate

1-n

apht

hol

Subs

trat

e ta

nnic

acid

Subs

trat

e gu

aiac

ol

(a)

(b)

(c)







4 Discussion



0

25

50

75

100

0 3 6 9 12

Enzy

me a

ctiv

ity (I

Um

L)

Time (days)

No lignin5gL lignin


0

25

50

75

100

0 3 6 9 12

Enzy

me a

ctiv

ity (I

Um

L)

Time (days)

0 120583M10 120583M20 120583M

30 120583M40 120583M50 120583M


0

25

50

75

100

0 3 6 9 12

Enzy

me a

ctiv

ity (I

Um

L)

Time (days)

pH 3pH 4pH 5

pH 6pH 7pH 8


0

25

50

75

100

0 5 10 15

Enzy

me a

ctiv

ity (I

Um

L)

Time (days)

20∘C30∘C

40∘C50∘C











66

43

29

14

(kDa)

1 2 1 2 1 2

(a) (b) (c)

Isozyme 1Isozyme 2

Isozyme 3




4is most frequently




119898 119881max and












1000

1500

2000

2500

3000

3500

0 5 10

Enzy

me a

ctiv

ity (I

Um

L)

pH

(a) pH optimum

2000

2500

3000

3500

4000

4500

0 20 40 60 80 100

Enzy

me a

ctiv

ity (I

Um

L)

Temperature (∘C)


0

1000

2000

3000

4000

0 50 100 150 200

Enzy

me a

ctiv

ity (I

Um

L)

Time (min)

pH 2pH 3pH 4pH 5

pH 6pH 7pH 8

(c) pH stability

0

1000

2000

3000

0 50 100 150 200

Enzy

me a

ctiv

ity (I

Um

L)

Time (min)

40∘C50∘C60∘C

70∘C80∘C






Competing Interests


References





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

25

50

75

100

0 3 6 9 12

Enzy

me a

ctiv

ity (I

Um

L)

Time (days)

No lignin5gL lignin


0

25

50

75

100

0 3 6 9 12

Enzy

me a

ctiv

ity (I

Um

L)

Time (days)

0 120583M10 120583M20 120583M

30 120583M40 120583M50 120583M


0

25

50

75

100

0 3 6 9 12

Enzy

me a

ctiv

ity (I

Um

L)

Time (days)

pH 3pH 4pH 5

pH 6pH 7pH 8


0

25

50

75

100

0 5 10 15

Enzy

me a

ctiv

ity (I

Um

L)

Time (days)

20∘C30∘C

40∘C50∘C











66

43

29

14

(kDa)

1 2 1 2 1 2

(a) (b) (c)

Isozyme 1Isozyme 2

Isozyme 3




4is most frequently




119898 119881max and












1000

1500

2000

2500

3000

3500

0 5 10

Enzy

me a

ctiv

ity (I

Um

L)

pH

(a) pH optimum

2000

2500

3000

3500

4000

4500

0 20 40 60 80 100

Enzy

me a

ctiv

ity (I

Um

L)

Temperature (∘C)


0

1000

2000

3000

4000

0 50 100 150 200

Enzy

me a

ctiv

ity (I

Um

L)

Time (min)

pH 2pH 3pH 4pH 5

pH 6pH 7pH 8

(c) pH stability

0

1000

2000

3000

0 50 100 150 200

Enzy

me a

ctiv

ity (I

Um

L)

Time (min)

40∘C50∘C60∘C

70∘C80∘C






Competing Interests


References





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


66

43

29

14

(kDa)

1 2 1 2 1 2

(a) (b) (c)

Isozyme 1Isozyme 2

Isozyme 3




4is most frequently




119898 119881max and












1000

1500

2000

2500

3000

3500

0 5 10

Enzy

me a

ctiv

ity (I

Um

L)

pH

(a) pH optimum

2000

2500

3000

3500

4000

4500

0 20 40 60 80 100

Enzy

me a

ctiv

ity (I

Um

L)

Temperature (∘C)


0

1000

2000

3000

4000

0 50 100 150 200

Enzy

me a

ctiv

ity (I

Um

L)

Time (min)

pH 2pH 3pH 4pH 5

pH 6pH 7pH 8

(c) pH stability

0

1000

2000

3000

0 50 100 150 200

Enzy

me a

ctiv

ity (I

Um

L)

Time (min)

40∘C50∘C60∘C

70∘C80∘C






Competing Interests


References





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





1000

1500

2000

2500

3000

3500

0 5 10

Enzy

me a

ctiv

ity (I

Um

L)

pH

(a) pH optimum

2000

2500

3000

3500

4000

4500

0 20 40 60 80 100

Enzy

me a

ctiv

ity (I

Um

L)

Temperature (∘C)


0

1000

2000

3000

4000

0 50 100 150 200

Enzy

me a

ctiv

ity (I

Um

L)

Time (min)

pH 2pH 3pH 4pH 5

pH 6pH 7pH 8

(c) pH stability

0

1000

2000

3000

0 50 100 150 200

Enzy

me a

ctiv

ity (I

Um

L)

Time (min)

40∘C50∘C60∘C

70∘C80∘C






Competing Interests


References





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Competing Interests


References





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

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