Chapter 4shodhganga.inflibnet.ac.in/bitstream/10603/8224/12/13_chapter 4.pdf · tested substrates were guaiacol > o-dianisidine > 1-napthol > pyrocatechol. Potential laccase inhibitor

Chapter 4

109

PURIFICATION AND CHARACTERIZATION OF LACCASE FROM

PLEUROTUS OSTREATUS IMI 395545

ABSTRACT

Extracellular laccase enzyme produced from Pleurotus ostreatus IMI 395545

was purified to homogeneity by ultrafiltration, ammonium sulfate precipitation, anion

exchange and size exclusion chromatography with a purification fold, yield and

specific activity of 4.8, 39.41% and 11.16 respectively. The strain produced two

laccase isoenzymes (LCC1 and LCC2) where LCC2 is the major isoenzyme produced

by the fungus. In the present investigation the laccase isoenzyme LCC2 was purified

to homogeneity and characterized. The purified laccase was a monomeric protein with

an apparent molecular mass of ~66 kDa. The optimum pH and temperature of the

LCC2 isoenzyme was found to be 6.0 and 60 °C respectively. LCC2 isoenzyme

showed maximum activity for 1 h at 65 °C and a half life of 3 h at the same

temperature. The kinetic parameters suggest that the order of the affinity towards the

tested substrates were guaiacol > o-dianisidine > 1-napthol > pyrocatechol. Potential

laccase inhibitor L-cysteine completely inhibited the activity at 0.1 mM.

Chapter 4

110

4.1. INTRODUCTION

Laccase is a multi-copper-containing enzyme catalyzing the oxidation of a

wide range of phenolic and aniline compounds. Because of its functional variety,

laccase has been purified from various sources, especially plants and fungi, and is

widely used for practical purposes. The catalysis carried out by all members of this

family is guaranteed by the occurrence of different copper centers in the enzyme

molecule [Baldrian, 2006]. The purification of a cell free laccase is an essential step

for the determination of accurate kinetic parameters due to the possible presence of

compounds from the host fungus that may act as natural mediators [Johannes and

Majcherczyk, 2000]. The most commonly used method for laccase purification is salt

elution from an anion-exchange resin, probably due to the higher stability of laccase

at neutral to slightly alkaline pH, as well as the pI of laccases (around 4.5).

Comparative studies of fungal laccases have shown that these enzymes are similar in

their specificity for different phenolic compounds, regardless of their origin, but differ

markedly in their inducibility, number of enzyme forms, molecular mass, redox

potentials, kinetic constants, substrate specificity, optimum pH and temperature [Xu

et al., 2000; Giatti et al., 2003; Palonen et al., 2003; Baldrian, 2004; Minussi et al.,

2007b; Park and Park, 2008]. Laccase is encoded by a family of genes and produced

in the form of multiple isozymes. It has been proven that genes encoding laccase

isozymes were differentially regulated [Soden and Dobson, 2001].

The substrate specificity of laccases varies from one organism to another. The

spectrum of laccase oxidizable substrates can be expanded considerably in the

presence of appropriate redox mediators [Johannes and Majcherczjk, 2000]. Due to

their interesting catalytic properties laccases have gained considerable interest in

various industrial areas. The ideal laccases for industrial use would exhibit stability at

Chapter 4

111

high temperature and pH conditions [Quaratino et al., 2007; Niladevi et al., 2008].

Wang et al. [2010b] reported a novel laccase with the property to tolerate cold

condition and high thermostable have characterized from Pycnoporus sp. Recently,

Several fungal laccase have been purified [Bryjak and Rekuc, 2010; Wong et al.,

2010; Pakhadnia et al., 2009] and many laccase have been purified and characterized

[Li et al., 2010; Sahay et al., 2009; Sahay et al., 2008].

In this chapter Pleurotus ostreatus IMI 395545 produced two extracellular

laccase isoenzymes (LCC1 and LCC2). The present study focused on the purification

and characterization of major extracellular laccase LCC2 isoenzyme.

4.2. MATERIALS AND METHODS

4.2.1. Chemicals

Acrylamide, ammonium persulfate, bis-acrylamide, Biogel P-200, coomassie

brilliant blue R-250, DEAE cellulose and TEMED (N,N,N’,N’- Tetramethyl

ethylenediamine) were purchased from s d fine-chem Limited, India. Guaiacol,

o-dianisidine, pyrocatechol and 1-napthol were purchased from LOBO (Cheme),

India. Protein marker was purchased from Heleni Biomolecules private limited,

Chennai, India. All other chemicals purchased were of analytical grade.

4.2.2. Laccase production

Laccase production was carried out in the optimized production medium in the

bioreactor ADI 1025 Bioconsole under optimized culture condition (kindly refer

chapter 3).

Chapter 4

112

4.2.3. Laccase assay

Laccase activity was determined using guaiacol as the substrate according to

the method of Sandhu and Arora [1985]. Kindly refer the first chapter for details

(1.2.6).

4.2.4. Estimation of Protein

The protein content of the culture filtrate was estimated by Lowry’s method

with bovine serum albumin as a standard [Lowry et al., 1951].

4.2.5. Extraction of laccase

Pleurotus ostreatus IMI 395545 was grown on the medium (Chapter 2) for ten

days. After ten days of growth the culture supernatant of the organism was filtered

through cheese cloth (4 fold) to remove mycelial debris. 1000 ml of culture

supernatant was centrifuged (6000 x g for 30 min) and filtered through Whatman

No. 1 to remove the fine particles.

4.2.6. Zymogram analysis of laccase on Native-PAGE

In order to determine the number of laccase isoenzymes produced by

Pleurotus ostreatus IMI 395545, the crude culture was centrifuged at 6000 x g for

30 min and the obtained supernatant was used for further studies. Native-PAGE was

carried out according to the method described by Gabriel [1971] using 5 mM guaiacol

in 100 mM sodium acetate buffer [pH 6.0] at room temperature [Das et al., 1997].

4.2.7. Laccase purification

The method for the laccase purification was adopted from a protocol described

by Das et al. [2001] with minor modifications. All operations were performed at 4 °C

Chapter 4

113

unless otherwise mentioned. The purification parameters calculations were carried out

according to Nelson and Cox [2004] (Appendix 2).

4.2.7.1. Ultrafiltration

The method for the ultrafiltration of laccase was adapted from a protocol

described by kim et al. [2002]. The extracted culture filtrate was concentrated by

ultrafiltration cell using Amicon 8200, YM-30 membrane through a membrane filter

(molecular weight cut off 10 kDa) until a 10-fold concentration was achieved.

4.2.7.2. Ammonium sulphate precipitation

Concentrated filtrate was brought to 40% (w/v) saturation ((NH4)2SO4,

overnight at 4 °C) then centrifuged at 6000 x g for 30 min. The obtained precipitated

pellet was then discarded. The resulting supernatant was brought to 80% (w/v)

saturation ((NH4)2SO4, overnight at 4 °C) then centrifuged at 6000 x g for 60 min at

4 °C. The precipitate was collected and then resuspended in 100 mM sodium

phosphate buffer pH 6.0 and dialyzed against the same buffer overnight at 4 °C. The

dialyzed enzyme sample was subjected to anion exchange chromatography.

4.2.7.3. Anion exchange column chromatography

The dialysate was loaded onto anion exchange (DEAE- cellulose) column

(22 x 220 mm) that had been pre-equilibrated with 100 mM sodium phosphate buffer

pH 6.0. The enzyme loaded column was washed with 500 ml of the same buffer to

remove unbound sample components. A step wise gradient system of NaCl (0.2-

1.0 M) in the 100 mM sodium acetate buffer [pH 6.0], was used to elute the bound

protein at a rate of 1ml/min; fractions were collected and assayed for laccase activity.

The active fractions of the laccase peaks were pooled together and dialyzed against

the same buffer.

Chapter 4

114

4.2.7.4. Size-exclusion chromatography

The dialysate was subjected to size exclusion chromatography in the column

(16 x 650 mm) packed with Biogel P-200, pre-equilibrated with 100 mM sodium

phosphate buffer pH 6.0. Active fraction were collected, assayed, pooled together and

dialyzed against same buffer. The dialysate was concentrated by lyophilizer (Mini

Lyodel Freeze Dryer, India) and stored at -20 °C for further characterization studies.

4.2.7.5. Determination of molecular mass

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)

was carried out according to the method of Laemmli [1970]. The same was used to

monitor the development of the purification process, to determine the homogeneity

and apparent molecular mass of the purified laccase. SDS-PAGE was carried out on a

4% w/v stacking gel and 10% w/v separating gel. The approximate molecular mass of

the laccase was determined by calibration against broad range molecular weight

markers, which contained the proteins β-galactosidase (116.25 kDa), phosphorylase B

(97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase

(30 kDa), soybean trypsin inhibitor (20 kDa) and lysozyme (14 kDa). SDS–PAGE

and native PAGE revealed the presence of two proteins. Non-denaturing PAGE was

performed to ascertain which protein correlated to laccase activity. The non-

denaturing gel was bisected and half was stained with Coomassie Brilliant Blue R-250,

the other half was stained with guaiacol to determine which band correlated to laccase

activity.

Chapter 4

115

4.2.8. Characterization of purified laccase

4.2.8.1. Effect of pH

The optimum pH of the purified laccase LCC2 isoenzyme was studied by

incubating the laccase over a pH range of 3.5-10.0. The buffer systems used were

100 mM sodium acetate buffer for pH 3.5-5.5; 100 mM sodium phosphate buffer

pH 6.0-8.0; 100 mM glycine – NaOH buffer pH 8.5-10.0. The purified laccase was

incubated at the above pH for 30 min and the residual activity was determined

spectrophotometrically at 470 nm by guaiacol as the substrate.

4.2.8.2. Effect of temperature

The temperature profile of the purified laccase was identified by incubating

the enzyme for 30 min at different temperatures from 20 to 80 °C with the increment

of 10 °C at the optimum pH determined previously. Thermostability of purified

laccase was determined by incubating the enzyme at temperature from 50 to 70 °C

with the increment of 5 °C for different time period (1-5 h). Residual activity was

determined spectrophotometrically at 470 nm using guaiacol as the substrate.

4.2.8.3. Kinetic constants

Kinetic constants of laccase for the most commonly used substrates guaiacol;

o-dianisidine, pyrocatechol and 1-napthol were investigated. The reactions were

conducted at standard assay condition. The wavelengths for laccase activity with the

above mentioned substrates were determined spectrophotometrically by allowing the

reaction of that substrate with laccase to proceed to completion, performing a spectral

scan and using suitable λmax (wavelength of maximum absorbtion). Kinetic studies

were conducted for the selected four substrates and the Vmax and Km values were

calculated using the Michaelis-Menten equation.

Chapter 4

116

4.2.8.4. Effect of inhibitors

The effects of several potential inhibitors were determined by incubating the

purified laccase with various concentrations of inhibitors and measured the residual

activity with guaiacol as substrate. L-cysteine, sodium metabisulphite, sodium

sulphite, sodium hydrogen sulphite and sodium dithionite was incubated with purified

laccase at four different concentrations (0.1, 2, 5 and 10 mM) for 30 min at room

temperature. The change in absorbance was measured spectrophotometrically at

470 nm. A control test was conducted in parallel in the absence of the inhibitor.

4.3. RESULTS

In order to identify the isoenzymes pattern of Plerurotus ostreatus IMI 395545,

crude laccase was subjected to native PAGE. This was followed by zymogram

analysis using guaiacol. The native PAGE results revealed that two laccase

isoenzymes (LCC1 and LCC2) were extracellularly produced by Pleurotus ostreatus

IMI 395545 (Figure 4.1).

Pleurotus ostreatus IMI 395545 laccase LCC2 isoenzyme was purified to

homogeneity from the culture filtrate using four step purification procedures as

summarized in table 4.1. In anion exchange chromatography DEAE-Cellulose column,

two enzyme peaks were eluted obtained by linear gradient elution. LCC1 and LCC2

were eluted at approximately 660 nm and 680 nm concentration of NaCl.

In ammonium sulfate precipitation, the specific activity was increased to 3.50 U/mg

protein and the yield was 65.8% with a purification factor of 1.44 fold. The dialyzed

sample was applied to a DEAE-cellulose column. In DEAE cellulose column

chromatography, the specific activity was increased to 7.16 U/mg protein and the

yield was 50.5% with purification factor of 2.95 fold.

Chapter 4

117

Fraction with major laccase (LCC2) activity were pooled and dialyzed without

any apparent loss of activity and loaded onto a Biogel P-200 column. At the end

of the purification process, LCC2 isoenzyme was purified to 4.8 fold with a yield of

39.41%. The purified LCC2 isoenzyme had a specific activity of 11.16 U/mg of

protein using guaiacol as substrate under standard assay condition. The purified

Pleurotus ostreatus laccase (LCC2) yielded a single band in SDS-PAGE after staining

with Coomassie brilliant blue R-250 and guaiacol, respectively (Figure 4.2). The

molecular weight of the LCC2 isoenzyme was calculated to be ~ 66 kDa.

The influence of pH within a range of 3.5 to 10 on laccase (LCC2) activity of

Pleurotus ostreatus IMI 395545 was studied and the results were plotted (Figure 4.3).

The optimum pH for the maximum laccase activity was found to be 6.0. The optimum

pH was not identical to the other substrates like o-dianisidine, 1-napthol and

pyrocatechol. The laccase shows optimum pH 5.5 for the substrates o-dianisidine and

1-napthol. The optimum pH for the pyrocatechol was at 5.0. When pH values greater

than 6.0, the enzyme activity decreased gradually and completely inactivated at higher

alkaline pH.

The residual activity of purified laccase LCC2 isoenzyme of Pleurotus

ostreatus IMI 395545 was determined at various temperatures (20-80 °C) using

guaiacol as substrate was depicted in the figure 4.4(A). The optimum temperature of

enzyme was found to be 60 °C. The enzyme showed highest activity between

55–65 °C. Beyond 65 °C the activity dropped sharply. The stability of the enzyme

with respect to temperature was also studied (Figure 4.4B). The Pleurotus ostreatus

IMI 395545 laccase LCC2 isoenzyme retained 100% of its initial activity after 3 h

incubation at 60 °C and 5 h incubation at 50 °C. Moreover the enzyme retains 80% of

Chapter 4

118

the maximum activity for 1 h at 65 °C. The laccase activity decreased rapidly when

incubated at 70 °C and the complete inactivation occurred within 0.5 h of incubation.

As mentioned in the methods, the purified laccase was characterized in terms

of its affinity constant (Km) and maximum velocity constant (Vmax) of the four

different substrates namely guaiacol, o-dianisidine, 1-napthol and pyrocatechol (Table

4.2). Reactions were initiated by addition of laccase and initial rates were obtained

from the linear portion of the progress curve. The fraction without enzyme served as

the control. The structure of the four substrates of the purified laccase (LCC2)

isoenzyme was shown in figure 4.5.

Effect of a range of potent laccase inhibitors on the laccase activity was tested

with guaiacol as substrate and the results are presented in table 4.3. Inhibitors were

added to the assay mixture at different concentrations. After incubation (30 min)

substrate was added and the residual enzyme activity was determined. Enzyme

inhibition was expressed in percentage.

4.4. DISCUSSION

More than one laccase isoenzyme, both constitutive and inducible, has been

detected in most white-rot fungi [Baldrian, 2006]. Two extracellular laccase

isoenzymes (LCC1 and LCC2) were secreted by Pleurotus ostreatus IMI 395545 as

shown in the figure 4.1. Mansur et al. [2003] reported that two laccase (LCC1 and

LCC2) was purified by simple purification steps like ammonium sulfate precipitation

DEAE and Biogel chromatography. Due to strong binding nature of laccase in the

DEAE gels from the same culture sample two more laccase (LCC3 and LCC4) were

purified by Isoelectric focusing native gels. The biochemical diversity of laccase

isoenzymes appears to be due to the multiplicity of laccase genes; however, regulation

Chapter 4

119

of their expression can be substantially diverse between fungal species [Palmieri et al.,

2003]. The presence of an inducer may result in the production of different isoforms

[Farnet et al., 1999; Palmieri et al., 2000; Pointing et al., 2000].

The yield of purified laccase isoenzyme (LCC2) from Pleurotus ostreatus IMI

395545 was 39.4% with purification fold of 4.8 (table 4.1). The yield of purified

laccase by this method was higher when compared to other reports in Pleurotus

species. Laccase purified from Pleurotus ostreatus strain V-184 by DEAE-Biogel

chromatography had a specific activity of 1883 U/mg, yield of 20.4% with the

purification of 11.8 fold [Mansur et al., 2003]. Laccase of the basidiomycete

Pleurotus florida was purified by ammonium sulfate precipitation followed by anion

exchange and Biogel P-200 chromatography had a purification fold of 34.32 with the

yield of 10.01% [Das et al., 2001].

The single band of purified laccase isoenzyme (LCC2) from the crude extract

was visualized by Coomassie Brilliant Blue, as shown in the figure 4.2 was calculated

to be ~66 kDa. Hublik and Schinner [2000] reported the laccase purified from

Pleurotus ostreatus is a monomeric protein with a molecular weight of 67 kDa. The

molecular weight of Pleurotus ostreatus D1 laccase proved to be approximately

64 kDa [Pozdnyakova et al., 2006]. As reported by Palmieri et al., [1993; 1997] the

molecular weights of laccases from other strains of the same fungus vary from 64 to

70 kDa. Our result is in good agreement with the earlier reports of many researchers.

Mansur et al. [2003] reported the molecular weight of purified laccase of Pleurotus

ostreatus to be 65 kDa.

The optimum pH for the LCC2 isoenzyme of Pleurotus ostreatus IMI 395545

laccase was found to be pH 6.0, which was quite similar to the of Pleurotus ostreatus

studied by Palmieri et al. [1997], Pleurotus pulmonarius [De souza and Peralta,

Chapter 4

120

2003] and Stereum ostrea [Viswanath et al., 2008] using guaiacol as the substrate.

Hublik and Schinner [2000] reported that laccase from Pleurotus ostreatus showed

the highest oxidation rate at pH 5.8, when syringaldazine was used as a substrate.

When the pH values higher than 6.0 the enzyme activity decreased gradually and

completely inactivated at higher alkaline pH (Figure 4.3). The pH activity profile of

laccase are often bell-shaped, with optima around 4 - 6, when measured with phenolic

substrates [Garzillo et al., 2001].

The temperature dependence of the LCC2 isoenzyme of Pleurotus ostreatus

IMI 395545 laccase activity is depicted in figure 4.4 (A). The optimum temperature of

enzyme was found to be 60 °C using guaiacol as substrate. The optimal temperature

of laccase can differ greatly from one strain to another. De souza and Peralta, [2003]

and Das et al. [2001] reported that Pleurotus pulmonarius and Pleurotus florida

laccases shows optimum at 50 °C. The laccase from Pleurotus ostreatus is almost

fully active in the temperature range of 40-60 °C, with maximum activity at 50 °C

[Palmieri et al., 1993]. Youn et al. [1995] contrarily reported that laccase from

Pleurotus ostreatus showed an optimum temperature between 30-35 °C. According to

Baldrian [2006] the optimum temperature of the Pleurotus ostreatus laccase varies

from 35-60 °C.

Temperature stabilities of laccases vary considerably, depending on the source

organism. In general, laccases are stable at 30-50 °C and rapidly lose activity at

temperatures above 60 °C [Palonen et al., 2003]. The purified laccase LCC2

isoenzyme was stable over a high range of temperature (3 h at 60 °C) and maintains

50% of the activity for 2 h at 65 °C temperature as shown in the figure 4.4(B). This

purified laccase LCC2 isoenzyme showed better thermostable property than earlier

reports. Palmieri et al. [1993] reported that Pleurotus ostreatus laccase was almost

Chapter 4

121

fully active in the temperature range of 40-60 °C and showed a half life of 30 min at

60 °C. The thermal stability of enzymes may be influenced by the presence of

hydrophobic or charged residues, which increase enzyme rigidity and restrict

conformational changes during substrate binding [Fields, 2001; Somero, 2004].

Laccases are considered to be non-specific to their substrates, being able to

oxidize a wide range of aromatic compounds and hence it is of interest in textile dye

bleaching, detoxification of contaminated soil and water. For this reason, in the

present work kinetics of laccase activity was studied with four different substrates

namely monomethoxy substituted phenolic substrate guaiacol, dihydroxy substituted

phenol compound o-dianisidine, 1-hydroxynapthalene and dihydroxy substituted

phenol substrate pyrocatechol. The main kinetic parameters, Vmax (maximum enzyme

velocity) and Km (affinity constant) were determined (Table 4.2). Purified laccase

shows highest activity towards guaiacol followed by substrates with order of

decreasing affinity were o-dianisidine, pyrocatechol and 1-napthol.

The enzyme shows strong affinity towards guaiacol (0.052 mM) when

compared with Km value of guaiacol (3.1 mM) reported by Palmieri et al. [1997]. Two

laccase isoenzymes purified from fruit bodies of Lentinula edodes shows very high

affinity of 0.917 mM for Lcc 1 and 0.350 mM Lcc 2 [Nagai et al., 2003]. Comparison

of the structure of the substrates and the affinity towards the enzyme (Km) is based

upon the number and the position of hydroxyl group and substitute methoxy in the

benzene ring (Figure 4.5). Highest affinity for guaiacol may be due to presence of

single hydroxyl and substituted methoxy group in benzene ring, next higher affinity is

o-dianisidine, it has two methoxy substituted group. While 1-napthol has single

hydroxyl group in the bulky napthalene ring may cause steric hindrance during the

Chapter 4

122

reaction. pyrocatechol has two hydroxyl groups in the benzene ring, which may cause

weak affinity towards the enzyme.

The optimum pH of the laccase differed with the substrate used. The optimum

pH for the guaiacol and the o-dianisidine was 6.0 and 5.5 respectively. The other two

substrates 1-napthol and pyrocatechol optimum activity lies at pH 5.0. The variation

of pH with respect to the substrate was reported by Palmieri et al. [1997] and Chernyk

et al. 2008. The highest activity of the Pleurotus ostreatus laccases with respect to pH

profile also varied with the changes of substrate. The variation of optimum pH might

due to different role of substrate protonation in the reaction mechanism [Palmieri et

al., 1993; Youn et al., 1995].

Table 4.3 shows the effect of chemical compounds on laccase LCC2

isoenzyme. L-cysteine completely inhibits the isoenzyme at 0.1 mM concentration.

Other thiol compounds like sodium hydrogen sulphite, sodium dithionite and sodium

metabisulphite inhibit the enzyme at 5 mM concentration and sodium sulphite needs

10mM concentration to completely inhibit the enzyme activity. Many sulfhydryl-

containing compounds, e.g. L-cysteine, sodium dithionite and sodium sulphite are

often referred to as laccase inhibitors. Lu et al. [2007a] and Baldrian [2004] reported

that L-cysteine is one of the effective inhibitor for fungal laccase. However, Johannes

and Majcherczyk [2000] showed that the observed inhibitory effect is actually caused

by the reduction of the oxidized substrate by the sulfhydryl compounds and not by

true inhibition of the enzyme. Laccase can be inhibited when the inhibitor binds

strongly to it stopping further catalysis of the reaction. This occurs when the Cu at the

catalytic center is removed/chelated or by competing for O2, which is the specific

co-substrate of laccase. Laccases have been known to be inhibited by diethyl

dithiocarbamate and thiogycolic acid probably due to their effect on copper at the

Chapter 4

123

catalytic centre of laccase and by several sulfhydryl compounds such as dithiothreitol,

thiogycolic acid, cysteine and diethyldithiocarbamic acid [Baldrian, 2006].

4.5. CONCLUSION

The main laccase isoform of Pleurotus ostreatus IMI 395545 (LCC2) was

purified to apparent electrophoretic homogeneity with 39.41% recovery and the

purification fold was 4.8. The purified enzyme exhibits narrow optimum pH and

temperature 6.0 and 60 °C respectively. The thermostable property and its wide range

of substrate oxidation, the purified laccase LCC2 isoenzyme may be considered as a

good choice for industrial applications. Because of the high yield and easy

purification procedure the LCC2 isoenzyme could be of interest for the

biotechnological applications that have been suggested for laccases from other fungal

species. The purified laccase isoenzyme (LCC2) has many desirable characteristics

such as abundant production, wide substrate oxidation and reasonable thermostable

property.

Chapter 4

124

Protein MW

Marker

1

Purified

sample

2

Purified

sample

3

66 kDa

45kDa

35kDa

20kDa

14kD

a

Figure 4.1. Zymogram analysis of laccase activity of Pleurotus ostreatus

IMI 395545 in native PAGE. Lane 1- Protein marker; Lane 2-

Zymogram analysis of laccase isoenzymes (LCC1 and LCC2)

on native-PAGE by guaiacol.

Figure 4.2. Molecular weight determination of purified laccase isoenzyme

(LCC2) on SDS-PAGE and zymogram analysis. Lane 1- Protein

marker; Lane 2- Purified laccase (LCC2); Lane 3- Zymogram analysis of purified laccase (LCC2).

31kDa

Lane 2 Lane 1

97kDa

116kDa

66kDa

45kDa

20kDa

14kDa

LCC2

LCC1

Chapter 4

125

0

20

40

60

80

100

120

3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10

pH

Res

idu

al a

ctiv

ity

(%)

Figure 4.3. The effect of pH on the activity of Pleurotus ostreatus IMI 395545

laccase (LCC2 ).

Figure 4.4. The effect of temperature on the activity of Pleurotus ostreatus IMI

395545 laccase (LCC2). (A) Optimum temperature

(B) Thermostability.

A

0

20

40

60

80

100

120

20 30 40 50 60 70 80

Temperature

Resi

du

al

acti

vit

y(%

)

(ºC)

0

0

20

40

60

80

100

120

1 2 3 4 5 6

Incubation period (h)

Res

idu

al a

ctiv

ity

(%

)

50 °C 55 °C 60 °C 65 °C 70°C

B

Chapter 4

126

OH

OCH 3

Guaiacol

OH

Pyrocatechol

OH

OH

1-Napthol

NH2

NH2

OCH 3

H3CO

o-dianisidine

Figure 4.5. The substrates of Pleurotus ostreatus IMI 395545 laccase (LCC2) .

Chapter 4

127

Table 4.1. Summary and purification procedure of Pleurotus ostreatus IMI

395545 extracellular laccases

Purification steps

Volume

(ml)

Total

laccase

activity

(U)

Total

protein

(mg)

Specific

activity of

laccase

(U/mg)

Purification

Fold

Yield

(%)

Crude enzyme

(culture filtrate)

1000 850 350 2.42 1.0 100

Ultrafiltration 100 780 240 3.25 1.34 91.7

Ammonium sulfate

precipitation (80%)

25 560 160 3.5 1.44 65.8

DEAE-Cellulose

LCC1 isoenzyme*

LCC2 isoenzyme

4

15

35

430

NE

60

NE

7.16

NE

2.95

4.1

50.5

Biogel P-200

LCC2 isoenzyme

10 335 30 11.16 4.8 39.41

*LCC1 isoenzymes was not part of the present study; NE = Not Estimated

Table 4.2. Kinetic parameters for Pleurotus ostreatus IMI 395545 laccase (LCC2)

Substrate Wave length pH Km (mM) Vmax (mM/Sec) Vmax/ Km

Guaiacol 470 6.0 0.052 2.86 55

o-dianisidine 450 5.5 0.083 1.53 18.4

1-napthol 425 5.0 0.125 0.94 7.52

Pyrocatechol 550 5.0 0.183 0.53 2.89

The enzyme activity assay was performed at 60 °C. All values were calculated by the

linear regression (correlation coefficient ≥0.98) of double reciprocal plots, 1/vo versus

1/[s], from every set of triplicate measurements.

116 kDa 66 kDa 45 kDa 35 kDa 20 kDa

Chapter 4

128

Table 4.3. Effect of inhibitors on Pleurotus ostreatus IMI 395545 laccase (LCC2)

Inhibitor Concentration (mM) Inhibition (%)

L-cysteine

0.1 100

2 100

5 100

10 100

Sodium sulphite

0.1 2

2 14

5 68

10 100

Sodium hydrogen sulphite

0.1 32

2 84

5 100

10 100

Sodium dithionite

0.1 75

2 96

5 100

10 100

Sodium metabisulphite

0.1 60

2 97

5 100

10 100

Values reported are the means of values from three independent experiments with a

maximal sample mean deviation of ± 5%.