PLEUROTUS OSTREATUS IMI

Transcription

1 PURIFICATION AND CHARACTERIZATION OF LACCASE FROM PLEUROTUS OSTREATUS IMI ABSTRACT Extracellular laccase enzyme produced from Pleurotus ostreatus IMI 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 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. 109

2 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 110

3 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 produced two extracellular laccase isoenzymes (LCC1 and LCC2). The present study focused on the purification and characterization of major extracellular laccase LCC2 isoenzyme MATERIALS AND METHODS 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 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). 111

4 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) 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] Extraction of laccase Pleurotus ostreatus IMI 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 ml of culture supernatant was centrifuged (6000 x g for 30 min) and filtered through Whatman No. 1 to remove the fine particles Zymogram analysis of laccase on Native-PAGE In order to determine the number of laccase isoenzymes produced by Pleurotus ostreatus IMI , 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] 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 112

5 unless otherwise mentioned. The purification parameters calculations were carried out according to Nelson and Cox [2004] (Appendix 2) 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 Ammonium sulphate precipitation Concentrated filtrate was brought to 40% (w/v) saturation ((NH 4 ) 2 SO 4, 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 ((NH 4 ) 2 SO 4, 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 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 ( 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. 113

6 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 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 ( 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 nondenaturing 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. 114

7 Characterization of purified laccase Effect of ph The optimum ph of the purified laccase LCC2 isoenzyme was studied by incubating the laccase over a ph range of The buffer systems used were 100 mm sodium acetate buffer for ph ; 100 mm sodium phosphate buffer ph ; 100 mm glycine NaOH buffer ph 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 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 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 λ max (wavelength of maximum absorbtion). Kinetic studies were conducted for the selected four substrates and the V max and K m values were calculated using the Michaelis-Menten equation. 115

8 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 RESULTS In order to identify the isoenzymes pattern of Plerurotus ostreatus IMI , 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 (Figure 4.1). Pleurotus ostreatus IMI 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. 116

9 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 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 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 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 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 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 117

10 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 (K m ) and maximum velocity constant (V max ) 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 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 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 118

11 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 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 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 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, 119

12 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 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 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 C. According to Baldrian [2006] the optimum temperature of the Pleurotus ostreatus laccase varies from C. Temperature stabilities of laccases vary considerably, depending on the source organism. In general, laccases are stable at 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 120

13 fully active in the temperature range of 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, V max (maximum enzyme velocity) and K m (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 K m 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 mm for Lcc 1 and mm Lcc 2 [Nagai et al., 2003]. Comparison of the structure of the substrates and the affinity towards the enzyme (K m ) 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 121

14 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 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 sulfhydrylcontaining 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 O 2, 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 122

15 catalytic centre of laccase and by several sulfhydryl compounds such as dithiothreitol, thiogycolic acid, cysteine and diethyldithiocarbamic acid [Baldrian, 2006] CONCLUSION The main laccase isoform of Pleurotus ostreatus IMI (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. 123

16 Lane 1 Lane 2 116kDa 97kDa 66kDa 45kDa LCC2 LCC1 31kDa 20kDa 14kDa Figure 4.1. Zymogram analysis of laccase activity of Pleurotus ostreatus IMI in native PAGE. Lane 1- Protein marker; Lane 2- Zymogram analysis of laccase isoenzymes (LCC1 and LCC2) on native-page by guaiacol. Protein MW Marker 1 Purified sample 2 Purified sample 3 66 kda 45kDa 35kD 20kD a 14kD 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). 124

17 Residual activity (%) Residual activity(%) Residual activity(%) Chapter ph Figure 4.3. The effect of ph on the activity of Pleurotus ostreatus IMI laccase (LCC2 ). A Temperature (ºC) B Incubation period (h) 50 C 55 C 60 C 65 C 70 C Figure 4.4. The effect of temperature on the activity of Pleurotus ostreatus IMI laccase (LCC2). (A) Optimum temperature (B) Thermostability. 125

18 OH NH 2 OCH 3 H 3 CO OCH 3 H 2 N Guaiacol o-dianisidine OH OH OH 1-Napthol Pyrocatechol Figure 4.5. The substrates of Pleurotus ostreatus IMI laccase (LCC2). 126

19 Table 4.1. Summary and purification procedure of Pleurotus ostreatus IMI extracellular laccases Purification steps Crude enzyme (culture filtrate) Total Specific Total Volume laccase activity of Purification Yield protein (ml) activity laccase Fold (%) (mg) (U) (U/mg) Ultrafiltration Ammonium sulfate precipitation (80%) DEAE-Cellulose LCC1 isoenzyme* 4 35 NE NE NE 4.1 LCC2 isoenzyme Biogel P-200 LCC2 isoenzyme *LCC1 isoenzymes was not part of the present study; NE = Not Estimated Table 4.2. Kinetic parameters for Pleurotus ostreatus IMI laccase (LCC2) Substrate Wave length ph K m (mm) V max (mm/sec) V max / K m Guaiacol o-dianisidine napthol Pyrocatechol 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/v o versus 1/[s], from every set of triplicate measurements. 127

20 Table 4.3. Effect of inhibitors on Pleurotus ostreatus IMI laccase (LCC2) Inhibitor Concentration (mm) Inhibition (%) L-cysteine Sodium sulphite Sodium hydrogen sulphite Sodium dithionite Sodium metabisulphite Values reported are the means of values from three independent experiments with a maximal sample mean deviation of ± 5%. 128