Characterization of tyrosinase and accompanying laccase ... - NOPR

Indian Journal of Biochemistry & Biophysics Vol. 40, February 2003, pp. 40-45

Characterization of tyrosinase and accompanying laccase from Amorphophallus campanulatus Pallavi S Paranjpe1, Meena S Karve2 and Subhash B Padhye1* 1

Department of Chemistry and 2Biochemistry, University of Pune, Pune 411007, India Received 13 March 2002; revised 22 November 2002

Tyrosinase and laccase activities were detected in the corm of Amorphophallus campanulatus after extraction with ethanol followed by ammonium sulphate precipitation (20-60%) and dialysis against 10 mM Na2HPO4 buffer at pH 7.0. Tyrosinase was found to be the predominant enzyme exhibiting mono- and di-phenolase activities, specificity for L-DOPA as substrate, optimum pH being 6.0, optimum temperature at 40°C and Km at 1.05 mM. Laccase showed substrate specificity for p-phenylenediamine (p-PD), Km at 2.7 mM, optimum pH being 5.0 and was inactivated above 40°C. Three isoforms of tyrosinase were detected on SDS-PAGE with apparent molecular mass ~127, 31 and 27 kDa respectively. On staining sections of A. campanulatus with L-DOPA as substrate and 3-methyl benzothiazolinone hydrazone (MBTH) for colour development, tyrosinase was detected in the intercellular spaces of the plant tissue. The cytosolic region did not show any colour indicating the absence of the enzyme.

The most abundant in terms of occurrence amongst the polyphenol oxidases (PPOs), is the enzyme tyrosinase (monophenol, o-diphenol: oxygen oxidoreductase, EC 1:14:18:1), while other enzymes falling under this class include laccase (p-diphenol: oxygen oxidoreductase, EC 1:10:3:2) and catechol oxidase (o-diphenol: oxygen oxidoreductase, EC 1:10:3:1) respectively1,2. In higher plants the PPOs are thought to be involved in the defense against insects and microorganisms by forming a resistant layer of melanin pigments on the surface of the damaged tissues, rendering it a brownish black colour3. This browning effect caused by the melanin compounds is undesirable in case of canned food products as it leads to the loss of food quality and market rejection4,5. On the other hand, it is found to be desirable in the production of tea, coffee and cocoa beverages for the development of colour and flavors6. Tyrosinases have been characterized from various plant sources4,7 and are known to utilize a variety of mono- and di-phenol substrates, although the preferred substrates for assaying the enzyme activity include tyrosine, dopa and catechol. The enzyme is distinguishable from _________ *Corresponding author E-mail: [email protected] Tel.: + 91 20 560 1227; Fax: +91 20 569 1728 Abbreviations used: L-DOPA: 3,4-dihydroxyphenylenediamine; SHAM: salicylhydroxymic acid; CTAB: cetyltrimethyl ammonium bromide; p-PD: p-phenylenediamine; MBTH: 3methyl-2-benzothiazolinone hydrazone; SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis; BSA: Bovine serum albumin; DMF: Dimethyl formamide.

other PPOs especially the laccase, using specific inhibitors such as SHAM, tropolone, cinnamic acid, 2,3-naphthalenediol, and 4-hexylresorcinol, which affect laccase activity minimally8. The tuberous roots of Amorphophallus campanulatus, commonly known as ‘Suran’are used in India in curries and pickles and are ascribed with therapeutic activities against piles and dysentery9. When the corm of this tuber is cut open it gets coated with a thick protective layer of brown pigment which is probably indicative of high levels of PPO activities associated with it. The tyrosinase extracted from this source has been successfully used in the fabrication of biosensors by us recently10,11. We describe here the extraction and characterization of tyrosinase from A. campanulatus along with the elucidation of the laccase activity associated with it, which contributes to the browning reaction and the histochemical localization of the tyrosinase. Materials and Methods Materials The corm of A. campanulatus was purchased freshly from the local market. The chemicals including L-DOPA, tyrosine, p-PD, MBTH and SHAM were obtained from Sigma Chemicals, UK. Catechol, 4-methyl catechol and CTAB were products of SRL Chemicals (India). The reagents for electrophoresis were procurred from BioRad (India). All other chemicals were of analytical grade.

PARANJPE et al.: CHARACTERIZATION OF TYROSINASE AND LACCASE FROM A. CAMPANULATUS

Preparation of enzyme Peeled corm of A. campanulatus (20 g) was homogenized in chilled 0.1 M Na2HPO4 buffer (pH 7.0) filtered through four layers of cheesecloth and was centrifuged at 2500 rpm for 7 min at 4°C. The insoluble matter was precipitated out from the supernatant by the addition of 12.5% (v/v) chilled ethanol and the supernatant was subjected to ammonium sulphate fractionation between 20 to 60% saturation at 4°C. The precipitate obtained at 60% saturation was re-suspended in the extraction buffer, dialyzed against 10 mM Na2HPO4 buffer (pH 7.0) and used as the enzyme source. To solubilize the membrane bound tyrosinase the supernatant after centrifugation (2500 rpm) was either repeatedly frozen at -70°C and thawed at room temperature or was incubated with 1% solution of Triton X-100 or SDS or Brij-35 overnight at 4°C, dialyzed against 10 mM Na2HPO4 buffer (pH 7.0) and assayed for the tyrosinase activity using L-DOPA as substrate12. Protein content of the isolated enzyme was determined by the method of Lowry et al.13 using BSA as standard. Enzyme assay Monophenolase activity The reaction mixture contained 4 mM tyrosine, 1.6 mM MBTH and 0.1 mM L-DOPA with 0.48 mg of enzyme solution in 1 ml of 0.1 mM Na2HPO4 buffer (pH 6.0) containing 2% DMF at 37°C. The enzyme activity was monitored spectrophotometrically at 505 nm14. Diphenolase activity The reaction mixture contained 0.48 mg of the enzyme solution in 1 ml of 0.1 mM Na2HPO4 buffer (pH 6.0) and different substrates such as 2 mM LDOPA (475 nm), 4 mM DL-DOPA (475 nm), 2 mM catechol (410 nm) and 4 mM 4-methyl catechol (410 nm), respectively12,15. The enzyme activity was measured spectrophotometrically at the wavelength indicated for each substrate. Laccase activity The reaction mixture contained 0.48 mg of the enzyme in 1ml of 0.1 M citrate-phosphate buffer (pH 5.0), 4 mM L-DOPA (475 nm) and 4mM p-PD (410 nm) as substrates respectively15. The enzyme activity was measured spectrophotometrically at the wavelength indicated for each substrate. The rates of

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reactions and km values were calculated from the linear region of absorbance versus time curves. The enzyme activity was defined in the unit of Katal, which is the amount of enzyme used in the conversion of one μmole of substrate per second. Optimum pH of tyrosinase and laccase Citrate-phosphate buffers (0.1 M) in the pH range of 4.0-6.0 and Na2HPO4 buffer in the pH range 5.8-7.5 were used. Tyrosinase activity was monitored using 2 mM L-DOPA and 4 mM tyrosine as substrates while laccase activity was measured employing 4 mM p-PD and L-DOPA as substrates respectively. Optimum temperature of tyrosinase and laccase The assay mixture containing 0.48 mg of the enzyme was incubated at different temperatures from 15-70°C for 5 min followed by the addition of respective substrate (2 mM L-DOPA for tyrosinase and 4 mM p-PD for laccase). Effect of inhibitors Effect of varying concentrations of the inhibitors on enzyme activity was studied. In the case of tyrosinase, SHAM (50-150 μM) was added to L-DOPA as substrate. In the case of laccase, CTAB (0.5-1.5 mM) was used as inhibitor and p-PD as substrate. The inhibition was measured as the decrease in the initial velocity. The effects of specific inhibitors like SHAM (for tyrosinase) and CTAB (for laccase) were determined using the final concentrations of 1 mM and 0.100 mM for SHAM and CTAB, respectively. The rate of reaction and % inhibition were calculated from the linear portion of the absorbance versus time plots. SDS-PAGE SDS-PAGE was carried out in 10% polyacrylamide gel in Tris-glycine buffer (pH 8.3) system by the method of Laemmli16 as modified by Angleton and Flurkey17 for the partially denaturing conditions. The sample was prepared in 0.1% SDS avoiding 2-mercaptoethanol and heat treatment. Tyrosinase was located by activity staining. After electrophoresis the gels were soaked in 10 mM Na2HPO4 buffer (pH 6.0) for 10 min and cut into vertical strips, followed by incubation in the same buffer containing 2 mM 18 L-DOPA and 4 mM MBTH at 37°C in the presence and absence of 150 μM of SHAM inhibitor. The isoenzyme patterns were recorded using Kodak film.

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INDIAN J. BIOCHEM. BIOPHYS., VOL. 40, FEBRUARY 2003

Histochemical studies Fresh corm sample was peeled and cut into the sections of 6 to 8 microns using rotary microtome (Richard Tung, UK) under ice-cold conditions. The sections were stained with L-DOPA and MBTH solutions in Na2HPO4 buffer (pH 6.0), just prior to the microscopic evaluation. Microphotographs were taken on Zeiss Axioptan microscopic assembly (Germany). Results and Discussion The major phenol oxidase in the enzyme extract of A. campanulatus was found to be tyrosinase although laccase activity was also observed. The simple extraction procedure employed helped to preserve both monophenolase and diphenolase activities in the enzyme extract. Loss of monophenolase activity during purification process involving removal of phenolics has been reported by some workers4,19. Monophenolase activity was found to be five and twelve times lower than laccase and diphenolase activity, respectively. Effect of substrates Tyrosinase activity in the corm extract was highest with L-DOPA as a substrate (Table 1). The apparent km value of 1.05 mM obtained (Fig. 1) from the Lineweaver-Burk double reciprocal plots of initial velocity against the substrate concentrations (L-DOPA) is similar to that reported for tyrosinases from other plant sources3. Laccase activity was higher with p-PD than with L-DOPA as substrate. The km value for p-PD was 2.7 mM and for dopa it was 28.5

mM, indicating that p-PD is the preferable substrate for laccase. Effect of inhibitors Tyrosinase and laccase activities were determined based on their selectivities towards ortho- and paradiphenoloxidases. Specific inhibitors, viz. SHAM and CTAB, were used as suggested by Kumar and Flurkey28. It was observed that ~12 μM SHAM competitively inhibited tyrosinase activity by 50%, L-DOPA being the substrate. CTAB (1 mM) caused mixed-type of inhibition on laccase activity resulting in 50% inhibition with p-PD as the substrate (Fig. 2). CTAB (1.5 mM at which laccase activity was inhibited more than 80%) had negligible effect on tyrosinase activity (Fig. 1). Effect of pH The optimum pH for tyrosinase was observed to be around 6.0 although stable activity was evident in the pH range of 6.0 to 7.0. The optimum pH for the laccase activity using p-PD and L-DOPA as Table 1—Substrate specificity of A. campanulatus tyrosinase [Activity is expressed as the amount of enzyme catalyzing the conversion of one μmole of substrate per sec] Substrate Activity (katal) k (mM) LDOPA 14010 1.05 DL-DOPA 2340 4.00 Catechol 5725 3.13 4-Methylcatechol 5150 5.26 Tyrosine 130 1.33

Fig. 1—Lineweaver-Burk double reciprocal plot of initial velocity against substrate concentration for the inhibition of A. campanulatus tyrosinase by (.), CTAB, 1.5 mM; (▲),SHAM, 100 μM; (□), No inhibitor [Reaction mixture included 0.48 mg protein, 2 mM L-DOPA, in 0.1 M Na2HPO4 buffer, pH 6.0]


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Fig. 2—Lineweaver-Burk plot for laccase activity with varying concentrations of CTAB inhibitor (□), No inhibitor; (.), CTAB, 0.5 mM; (▲), 1.0 mM; (●) 1.5 mM [Ki values were calculated from the X- and Y-axis intercept using the equation: Y intercept = 1+ ([I]/α Ki)/vmax; X intercept = 1+ ([I]/α Ki)/Km (1+ [I]/ Ki). The data is fitted in a linear regression plot]

substrates was 5.0 and 7.0, respectively (Fig. 3). These are consistent with the pH profiles reported for fungal laccases20. There was no lag period for monophenolase activity at pH 6.0, using L-tyrosine as the substrate in the presence of catalytic amount of L-DOPA (0.1 mM). The activity was found to increase with increase in pH up to 7.0 after which it dropped considerably giving a bell-shaped curve (Fig. 4). This observation is consistent with the properties reported for monophenolase activity of grape polyphenol oxidase by Sanchez-Ferrer et al.21. Effect of temperature As shown in Fig. 5, the enzyme extract of A. campanulatus showed minor losses in the tyrosinase activity even at 45 to 50°C. Laccase activity underwent rapid inactivation when preincubated for 5 min at temperatures > 40°C. The energy of activation for tyrosinase was found to be Ea = 11,502 cal/mole at 40°C. The energy of activation for denaturation of 50% of tyrosinase activity was obtained as Ea = 7,145 cal/mole at 60°C. SDS-PAGE Partially denaturing SDS-PAGE of the enzyme extract of A. campanulatus showed three isoforms (Fig. 6) with Rf values of 0.04, 0.54 and 0.59 respectively on staining with L-DOPA and MBTH. A parallel gel was stained with Coomassie Blue R250

Fig. 3—Effect of pH on (a) tyrosinase and (b) laccase activities [The reaction mixture included (a), for tyrosinase: 0.48 mg protein, and 2 mM L-DOPA (●) (b), for laccase: 0.48 mg protein, 4 mM p-PD (○) or 4 mM L-DOPA (●) in 0.1 mM citratephosphate buffer, pH 4-6 and Na2HPO4 buffer, pH 5.8-7.5]

for protein detection. Apparent molecular masses of the isoforms calculated by the method of Weber and Osborn22 were found to be ~127, 31 and 27 kDa, respectively. The enzyme extract contained both tyrosinase and laccase activities, wherein the former could be completely inhibited by SHAM. In order to differentiate between tyrosinase and laccase isoforms after the electrophoretic run, the gels were stained for the tyrosinase activity in the presence of SHAM

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INDIAN J. BIOCHEM. BIOPHYS., VOL. 40, FEBRUARY 2003

Fig. 4—Monophenolase activity of A. campanulatus tyrosinase using L-tyrosine as substrate [The reaction mixture consisted of 0.48 mg protein, 4 mM tyrosine, 1.6 mM MBTH and 2% DMF in 0.1 mM Na2HPO4 buffer, pH 6.0. In the presence of catalytic amount of L-DOPA the characteristic lag period was completely abolished. [The insert shows monophenolase activity as a function of pH. Data fitted by a non-linear regression analysis]

Fig. 5—Effect of temperature on the initial velocity of tyrosinase and laccase activities of A. campanulatus [Details are mentioned under Materials and Methods]

(Fig. 6a) which revealed only one band with less color intensity and of Rf value, 0.48. This indicated that isoforms with Rf values 0.04, 0.54 and 0.59 were completely inhibited by SHAM and correspond to tyrosinase, while the band having Rf of 0.48 represented the laccase activity. Histochemical studies In order to localize the tyrosinase enzyme in situ, sections of A. campanulatus were stained with the

Fig. 6—Detection of the 3 isoforms of tyrosinase and of laccase by staining SDS-PAGE gel strips [Staining by L-DOPA and MBTH solution (a), in presence of 150 μM SHAM yielded a faint band due to laccase; (b), in the absence of SHAM yielded 3 bands due to the isoforms of tyrosinase. Commassie Blue protein staining was carried out for (c), enzyme sample and (d), molecular weight marker (Arrow indicates the faint band). Details are described under Materials and Methods]

specific substrate (i.e. L-DOPA) and scanned microscopically. Figs 7a and b show the microscopic pictures of these sections before and after staining with L-DOPA and MBTH solutions. It was observed that L-DOPA reacted with tyrosinase in situ forming the dopaquinone intermediate, which instantaneously reacted with MBTH forming a pink colored complex at the site of the enzyme. Tyrosinase was thus found


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References

Fig. 7—In situ detection of A. campanulatus tyrosinase [A: sections before staining; B: Sections after staining for tyrosinase-specific activity using L-DOPA and MBTH solution as described under Materials and Methods; Magnification power, 400 units]

to be localized in the intercellular spaces of the plant tissue, probably attached to the cell wall membrane. The staining did not appear continuously throughout the tissue but appeared as patches. When monitored microscopically over a period of time, the cytosolic region of the cell remained colourless indicating the absence of the enzyme. Acknowledgement PP would like to thank British Council for a visitorship program at UMIST, Manchester (UK) and Dr Manoj Mojamdar, formerly of National Center for Cell Science, Pune, for his keen interest in the present work. SP would like to acknowledge cooperation and help received from Professor William Flurkey (Indiana State Univ., Terre Haute, USA) in confirming some of the experimental results. The authors would like to thank Ms. Sabari Dutta for the technical help provided in preparation of this manuscript.

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