Biosci. Biotech. Res. Comm. 8(2): 161-170 (2015)

Comparative evaluation of puri•ed and characterized tyrosinases from two edible mushrooms, Agaricus bisporus and Pleurotus ostreatus and their clinical potential

Kamal U. Zaidi1 and Ayesha S. Ali*

1Biotechnology Pharmacology Laboratory, Centre for Scienti•c Research & Development, People’s University Bhopal-462037, India and Department of Zoology & Biotechnology, Sai•a College of Science, Bhopal-462037, India


Since the discovery of the melanogenic properties, tyrosinase has been in prime focus and microbial sources of the enzyme are sought. Agaricus bisporus and pleurotus ostreatus widely known as the common edible mushroom due to its high amounts of proteins, enzyme, carbohydrates, !bers, and low fat contents which are frequently cited in the literature in relation to their nutritional values. In the present work, comparative analysis was done for tyrosi- nase recovered from A. bisporus and P. ostreatus. The enzyme was puri!ed by ammonium sulphate precipitation, dialysis followed by gel !ltration chromatography on Sephadex G-100, and ion exchange chromatography on DEAE- Cellulose; the enzyme of A. bisporus was puri!ed, 16.36-fold to give 26.6% yield on total activity in the crude extract and !nal speci!c activity of 52.19U/mg and puri!ed enzyme of P. osreatus showed a speci!c activity of 46.4 U/mg with 20.3 % yield. The SDS-PAGE electrophoresis showed a migrating protein band molecular weight of 95 kDa and 75 kDa for A. bisporus and P. ostreatus respectively. The puri!ed tyrosinase was optimized with the optimum values at pH7.0 and 6.0, temperature at 35ºC. The highest activity was reported towards its natural substrate, L-DOPA, with an apparent Km value of 0.933 mM and 0.119 mM of puri!ed enzyme of A. bisporus against P. ostreatus. This indi- cated that tyrosinase puri!ed from Agaricus bisporus is a potential source for medical applications.



*Corresponding Author: Received 2nd October, 2015

Accepted after revision 15th November, 2015 BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007 NAAS Journal Score : 3.48

© A Society of Science and Nature Publication, 2015. All rights161 reserved.

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Kamal U. Zaidi and Ayesha S. Ali


Enzymatic browning results from the action of a group of enzymes namely tyrosinase. This enzyme is widely distributed in nature, including bacteria, fungi, higher plants (with particularly high amounts in mushroom, banana, apple, pear, potato, avocado, and peach), and animals (Mayer, 2006). Tyrosinase performs a variety of functions in these organisms ranging from pigmentation to defense to sclerotization. The enzyme catalyzes at least two different reactions using phenolic compounds and molecular oxygen as substrates.These reactions include hydroxylation of monophenols to form ortho-diphenols (monophenolase or cresolase activity) and oxidation of diphenols to form ortho - diquinones (diphenolase or catechol oxidase activity). It has also been suggested that tyrosinase can oxidize 5,6- dihydroxyindole to the 5,6-dihydroxyquinone,(Korner and Pawelek, 1982).

There are several oxidases that can use phenolic sub- strates present in mushrooms, but tyrosinase is probably the principal enzyme involved in browning reactions in Agaricus bisporus (Jolivet et al., 1998). For exam- ple, tyrosinase, laccase and peroxidase were detected in Portabella mushrooms, a brown strain of Agaricus bisporus, but tyrosinase was present in larger amounts than either laccase or peroxidase (Zhang et al., 1999). The conversion of phenols to o-diphenols by tyrosinase is a potentially attractive catalytic ability and thus tyro- sinase has attracted a lot of attention with respect to its biotechnological applications; catechol products are useful as drugs or drug synthons, e.g. L- dopa (Claus and Decker, 2006). The physiological role of tyrosinase is related to melanin biosynthesis, especially in fungi, (Zaidi et al., 2013; Zaidi et al., 2014a, b).

In fungi, melanins are involved in defense mecha- nisms against stress factors such as UV or gamma radia- tion, free radicals, dehydration and extreme tempera- tures (Halaouli et al., 2006). The stability of fungal spores also bene!ts from the protective role of melanins (Mayer and Harel, 1979). In addition, tyrosinases are associated with wound healing, immune response in plants (Muller et al., 2004) and with sclerotization of the cuticle in insects (Terwilliger,1999). Mushroom tyrosinases have been the source of much biochemical interest in recent years due to the fact that they show a wide range of biological activities. Much of their activities are due to their melanogenic properties with metal ions. On review- ing the literature, it becomes evident that many species of mushrooms such as Lentinula edodes (Kanda et al., 1996), Amanita muscaria (Muller et al., 1996), Pycnop- orus sanguineus (Halaouli et al., 2005) and Lentinula boryana (Faria et al., 2007) have been used to extract tyrosinase. The present study has focused on compara- tive analysis on puri!cation and characterization of

tyrosinase from A. bisporus and P. ostreatus for the !rst time. For the best of our knowledge the puri!ed tyrosi- nase showed very high similarities to the other sources of tyrosinase, and have tremendous clinical potential.



Extraction of mushroom tyrosinase was performed by the method of Haghbeen et al., (2004), with few modi- !cations. The sliced mushrooms were homogenized by waring blender. Enzyme extraction was prepared with 500mL of cold 100mM phosphate buffer (pH 5.8) for 300g of mushroom. The homogenate was centrifuged at 5000 rpm for 30min and supernatant was collected. The sediments were mixed with cold phosphate buffer and were allowed to stand in cold condition with occasional shaking. Then the sediment containing buffer was sub- jected to centrifugation once again to collect superna- tant. The supernatant was used as a source of enzyme.


The puri!cation of tyrosinase was performed by the method of Haghbeen et al., (2004), with minor modi!ca- tion. Crude enzyme extract puri!ed by salt precipitation, dialysis, gel !ltration, ion exchange chromatography, and so forth has been employed in series so as to obtain the enzyme in its purest form. The pure enzyme thus produced can be used for the further analysis.


Ammonium sulphate precipitation was done in an ice bath using the !nely grounded ammonium sulfate. The powder was weighed and added slowly to the extract by constant stirring to ensure complete solubility, and the solution was centrifuged at 5000 rpm for 30 min at 4ºC. Different precipitation steps were carried out for tyrosinase enzyme precipitation (45– 80%) and precipi- tates were collected. The precipitate was dialyzed against 100mMpotassiumphosphatebuffer (pH7.0) for 24 h by changing the buffer thrice. The dialyzed fraction was used for tyrosinase activity and protein content.


The tyrosinase activity assay was performed as reported by Sung and Cho, (1992) spectrophotometrically, meas- uring conversion of L-DOPA to red colored oxidation

product dopachrome. The initial rate of reaction is pro- portional to concentration of the enzyme. An aliquot containing tyrosinase was incubated for 5min at 35ºC at time zero, 1mL of L-DOPA solution (4mg/mL) for measured at 475 nm. After incubation for additional 5min, the mixture was shaken again and a second read- ing was determined and was measured for 3 minutes. The change in absorbance was proportional to enzyme concentration. One unit of enzyme corresponded to the amount which catalyzed the transformation of 1 μmol of substrate to product per min under the above condi- tions and produced 1.35 changes in absorbance. Speci!c activity was expressed as enzyme unit per milligram of protein. The protein content of the enzyme was deter- mined by the method of Lowry et al., 1951 with bovine serum albumin as standard.


The dialyzed ammonium sulfate fraction was applied to a Sephadex G-100 column that was preequilibrated with a 100mM phosphate buffer of pH 7.0.The protein elution was done with the same buffer at a #ow rate of 5 mL/ min. The fractions were collected at 4º C. It was assayed for protein at 280nm as well as for enzyme activity. The active fractions were pooled, dialyzed against the 100mM phosphate buffer of pH 7.0, and concentrated.



Dialyzed enzyme preparation obtained after ammo- nium sulphate precipitation and Sephadex G-100 col- umn was subjected to ion exchange chromatography using DEAE-Cellulose column (20 × 1 cm). The dialyzed enzyme preparation was loaded on DEAE-Cellulose col- umn which was preequilibrated with potassium phos- phate buffer (100mM, pH 7.0).The column was washed !rst with equilibrated buffer and then bound proteins were eluted using linear gradient of 0–100mM NaCl and 0–100mM potassium phosphate buffer at a #ow rate of 1mL per min. The fractions (2.5mL each) were collected and assayed for tyrosinase activity and those showing high activity were pooled and used for SDS-PAGE anal- ysis.


SDS-PAGE was performed using a 12% separating gel and 4% stacking gel. The samples were heated for 5min at 100ºC in capped vials with 1% (w/v) SDS in the presence of -mercaptoethanol. Electrophoresis was

Kamal U. Zaidi and Ayesha S. Ali

performed at a 125V for 4 h in Tris- HCl buffer of pH

8.3.After electrophoresis, proteins in the separating gel were made visible by staining with Coomassie Brilliant Blue R-250.The standards used to make a plot of log molecular weight versus mobility of the protein band were lysozyme (20 kDa), myoglobin (26 kDa), carbonic anhydrase (38 kDa), ovalbumin (46 kDa), glutamate (62 kDa), bovine serum albumin (91 kDa), -galactosidase (120 kDa), and myosin (200 kDa).


The activity of tyrosinase was evaluated at different pH values in the range between pH 3 and 10 under assay conditions and the amount of dopachrome was deter- mined. Buffers used were citrate phosphate (pH 3.0–5.0), potassium phosphate (pH 6.0-7.0), Tris-HCl (pH 8.0-9.0), and glycine-NaOH (pH 9.0-10). Optimum temperature for enzyme activity was determined by incubating the standard reaction mixture at temperatures ranging from 35 to 65ºC.


The enzyme kinetics as measured by the Michaelis con- stant (Km) is de!ned as the substrate concentration at half the maximum velocity, the rate of enzymatic reac- tions, by relating reaction rate to the concentration of a substrate. The Michaelis constant (Km) value of the puri!ed enzyme was estimated in a range of tyrosinase concentrations. The apparent Km value of puri!ed tyro- sinase was calculated from the Lineweaver-Burk plots relating 1/V to 1/[S].


The partial puri!cation of tyrosinase, the 400 mL crude extract was obtained after extraction of A. bisporus and the speci!c activity of tyrosinase was observed 3.189 U/ mg of protein and protein content was 321.14 mg with 100% yield and 1 fold of puri!cation. On extraction of P. ostreatus, 300 mL crude extract was obtained which showed speci!c activity 2.83 U/mg of protein and pro- tein content was 274 mg with 100% yield and 1 fold puri!cation. Isolation of tyrosinase was most effective with ammonium sulphate precipitation. 70% concen- tration of this salt gave a precipitate rich in tyrosinase activity (856U/mL). After fractionation with ammonium sulphate (70% saturation) of A. bisporus the speci!c activity of tyrosinase was increased from 3.189 to 11.09 U/mg. The ammonium sulphate fractions were subjected to a concentrate, which resulted 77.13 mg of protein with 83.5% yield and 3.47 fold puri!cation (Table1).

Kamal U. Zaidi and Ayesha S. Ali

Table 1: Puri!cation of tyrosinase from edible mushrooms

In P. ostreatus the speci!c activity of tyrosinase got increased from 2.83 to 9.90 U/mg. The ammonium sul- phate fractions were concentrated which resulted 50.08 mg of protein with 64.7% yield and 3.49 fold puri!ca- tion (Table1). After the 70% ammonium sulphate precip- itation the speci!c activity of A. bisporus was 11.09 U/ mg, 77.13 mg of protein with 83.5% yield and 3.47 fold puri!cation. Although, the speci!c activity of P. ostrea- tus was 9.90 U/mg, 50.08 mg of protein with 64.7% yield and 3.49 fold puri!cation which is lower than A. bisporus tyrosinase. The previous !ndings were identi- cal to that reported by Lee et al., (1997), who found that 70% ammonium sulfate was the best fraction which gave the highest yield of tyrosinase activity from Sola- num melongena.

After fractionation with dialysis of Agaricus bisporus the speci!c activity of tyrosinase was increased from

11.09to 14.71 U/mg. The dialysed fractions were sub- jected to concentrate which resulted 33.63 mg of protein with 48.2% yield and 4.61 fold puri!cation (Table1).

In Pleurotus ostreatus the speci!c activity of tyro- sinase was increased from 9.90 to 11.3 U/mg. The dia- lysed fractions were concentrated which resulted 25 mg of protein with 36% yield and 3.99 fold of puri!cation (Table1). After the Dialysis the speci!c activity of A. bis- porus was 14.71 U/mg, 33.63 mg of protein with 48.2% yield and 4.61 fold puri!cation. While, the speci!c activity of P. ostreatus was 11.3 U/mg, 25 mg of pro- tein with 36% yield and 3.99 fold of puri!cation which is lower than A. bisporus tyrosinase. The concentrated protein obtained after dialysis was then subjected to further puri!cation using molecular sieving. Molecular !ltration of partially puri!ed tyrosinase was performed in Sephadex G-100 column .By the elution pro!le of gel

!ltration chromatography of Agaricus bisporus it was observed that the enzyme eluted major peaks of tyro- sinase activity which were in active fractions resulted 9.22-fold puri!cation with a !nal speci!c activity of

29.42U/mg. The overall recovery of the puri!cation was 35.7% (Figure 1a, Table 1). In Pleurotus ostreatus the collected proteins of dialysed ammonium sulfate saturated fractions were chromatographed on Sephadex G-100 column which showed that the pro!le fractions contained different protein molecules, although only one peak showed activity for tyrosinase. In this step the speci!c activity of the puri!ed tyrosinase was found to be 22.8 U/mg, 24.9% yield and 8.05 fold puri!cation. The active fractions were pooled, dialyzed and !nally concentrated (Figure 1b, Table 1).

After the Sephadex G-100 column chromatography the active fraction of A. bisporus was found 9.22-fold puri!cation with a !nal speci!c activity of 29.42 U/ mg. The overall recovery of the puri!cation was 35.7%. Although, the speci!c activity of the puri!ed tyrosi- nase of P.ostreatus was found to be 22.8 U/mg, 24.9% yield and 8.05 fold puri!cation which is quite lower than A. bisporus tyrosinase. The data reported by other researchers regarding the speci!c activity of tyrosinase, isolated from different species of mushrooms was found to be highly variable, Trifolium pretense as 5.94 U/ mg (Schmitz et al., 2008), Crocus sativus as 27 U/mg (Saiedian et al., 2007), Agaricus bisporus as 30 U/mg (Shi et al., 2002), Pycnoporus sanguineus as 30.2 U/mg (Halaouli et al., 2005) and Aeromonas media as 34U/mg (Wan et al., 2009). Horowitz et al.,(1970) reported that tyrosinase that is produced in the fruiting body can be recuperated and puri!ed by homogenizing in a blender and then passed through a French press followed by

Kamal U. Zaidi and Ayesha S. Ali

FIGURE 1: Elution pro!le of Gel !ltration chromatography of tyrosinase of A. bisporus [A] and P. ostreatus [B] on Sephadex G-100 column. Total protein was monitored at 660 nm and the fractions were assayed for the tyrosinase activity at 475nm.

acetone or ammonium sulfate precipitation (Mueller et al., 1996). The resuspended precipitate was further puri- !ed by one or more chromatography columns. The most commonly used columns are hydroxylapatite (Bouch- illoux, et al., 1963), DEAE-Cellulose (Fan et al., 2004) or DEAE Sepharose (Halaouli et al., 2005), various other immunoaf!nity resins (Khan et al., 2005), and Sephadex size exclusion gel

Utilization of Ion exchange chromatography resulted in excellent puri!cation index. The concentrated protein was dissolved in 100mM phosphate buffer of pH 7.0 and was applied on DEAE-cellulose column and was eluted with discontinuous gradient of NaCl 1to100mM (Figure 2a). The eluted active fractions rechromatographed on the same column with a linear gradient of potassium phosphate buffer (0–100mM) were passed through the column. This two-step puri!cation scheme ion exchange chromatography, resulted in a partially puri!ed tyrosi- nase preparation, obtained by pooling fractions 25, 26, 27, and 28 and the enzyme was puri!ed by about 16.36- fold puri!cation with a !nal speci!c activity of 52.19U/ mg. The overall recovery of the puri!cation was 26.6% (Figure 2b, Table 1). In Pleurotus ostreatus, the further

puri!cation of the enzyme rich fractions of the Sepha- dex G-100 gel !ltration was done using DEAE cellulose column. A sharp distinctive peak of tyrosinase activity, which !ts under one protein peak only, was obtained by pooling fraction 16. In this step the speci!c activity of the puri!ed tyrosinase was found to be 46.4 U/mg, 20.3% yield and 16.39 fold puri!cation was achieved. The active fractions were pooled, dialyzed and !nally concentrated (Figure 2c, Table 1).


Protein fractions after each step of puri!cation, viz., ammonium sulphate fractionation, dialysis, Sephadex G-100 gel !ltration chromatography and DEAE-cel- lulose chromatography along with crude extract were analyzed by SDS-PAGE. From the electrophoretic pat- tern it is evident that the crude and ammonium sul- phate fractionations contained many protein bands and were not properly resolved. Sephadex G-100 gel !ltra- tion chromatographic fraction showed distinct protein

Kamal U. Zaidi and Ayesha S. Ali

FIGURE 2: Elution pro!le of DEAE-Cellulose column chromatography of A.bisporus eluted by NaCl (0–100mM) [A]eluted by PBS (0–100mM) [B] and P.ostreatus [C] An aliquot of each fraction was assayed for protein content and tyrosinase activity.

bands, whereas DEAE-cellulose ion exchange chroma- tography showed a single protein band correspond- ing to single peak of enzyme activity observed in ion exchange elution pro!le. It indicated that protein was puri!ed to apparent homogeneity and is composed of only a single polypeptide chain. The molecular weight of the puri!ed protein was determined by interpolation from a semi-logarithmic plot of relative masses versus the Rf values depending on the relative mobility. SDS- PAGE of the tyrosinase of A. bisporus preparation from different puri!cation steps showed that the resolved electrophoretic bands were progressively improved from the crude extract to the !nal step of the DEAE-Cellulose column.

It revealed only a single distinctive protein band for the pure preparation of tyrosinase with an apparent molecular weight of ~94 kDa (Figure 3a). In this respect, tyrosinase puri!ed from Aspergillus oryzae, Trichode- rma reesei, and Aspergillus nidulans was with smaller molecular weight in the range of 67, 73.2, and 80.48 kDa (Ichishima et al., 1984; Selinheimo, et al., 2006; Birse et al., 1990). Kanda et al., 1996 obtained two activity peaks after ion exchange chromatography of an extract

from Lentinula edodes. When the fractions correspond- ing to each peak were analyzed by partially denaturing SDS-PAGE, both had three bands that showed tyrosinase activity. Fully denaturing SDS-PAGE of the same frac- tions gave bands at 15, 49, and 54 kDa for one fraction and 15, 50, and 55 kDa for the other puri!ed tyrosinase from Lentinula edodes (Kanda et al., 1996) and exhibited a molecular weight of 105 kDa.

SDS-PAGE of tyrosinase of P. ostreatus showed that the puri!cation scheme removed most of the major pro- teins with lower molecular weight found in the crude extracts (Figure 3b lane A). One major stained band of protein was present in DEAE-Cellulose fraction along with a protein with less staining intensity. The esti- mated size of the major bands was approximately ~75 kDa (Figure 3b lane E). The molecular weight of tyrosi- nase was found to be highly variable with various stud- ies conducted by other researchers. Similar results have been reported from the fungal tyrosinase of Aspergilus orizae being 67 kDa (Ichishima et al., 1984), of Lentinula edodes being 70 kDa(Kanda et al., 1996) and of the Pro- tabella mushroomas70 kDa (Fan and Flurkey, 2004). Comparative assessment on the molecular weight of

FIGURE 3: Gel electrophoresis of tyrosinase of P. ostrea-

tus and A. bisporus Lane A: standard protein of different

molecular weight; Lane B: crude extract; Lane C: ammo-

nium sulfate fraction; Lane D: dialysis; Lane E: Sephadex

G-100 gel !ltration fraction; and Lane F: DEAE-Cellulose fraction. Arrow (π) indicates location of tyrosinase approximately ~75 kDa [A] ~94 kDa [B].

Kamal U. Zaidi and Ayesha S. Ali

some plant tyrosinases lies approximately between 40 to 65 kDa as in Brassica oleracea (Gawlik et al., 2007) and Trifolium pretense (Schmitz et al., 2008). These reports have simiarity with the present study



Tyrosinases with various physicochemical features have been reported from various organisms. These enzymes generally have a pH optimum in the neutral or slightly acidic range. The tyrosinase from T. reesei and I. batatas has a basic pH optimum of 9 and 8, respectively (Selin- heimo et al., 2006; Eicken et al., 1998). Results (Figure 4) revealed that pH 7.0 was the optimal pH for tyrosinase from A. bisporus using phosphate buffer. These results coincide with that of Liu et al,. 2005, who reported that the maximal tyrosinase activity of Bacillus megaterium was 7.0, and the optimal L-tyrosinase activity extracted from Trichoderma reesei was 9.0 (Selinheimo et al., 2006). The pH-dependent changes in the kinetic properties of the mushroom tyrosinase are similar to the pH-dependent changes in the kinetic properties of tyrosinase from B-16 murine melanoma and human skin and thus appear to be a general property of tyrosinase from diverse sources. Our results also demonstrated that tyrosinase retained about 65 % of its activity after storing at pH 7.0 for 24 h. This means that tyrosinase of A. bisporus had higher pH stability over a wide range of pH values. The effects of pH on L-DOPA oxidation by the tyrosinase are shown in (Figure.4). The optimum activity of the tyrosinase from P.ostreatus on L-DOPA was observed at pH 6.0 whereas from pH 4.0 to 5.0, the enzyme had 20 to 30% activity.

FIGURE 4: Effect of pH on tyrosinase activity of the crude extract of P. ostreatus and A. bisporus. Assays were performed at 35˚C and data were obtained as mean value of optical density. The optimum activity of the P. ostreatus tyrosinase at pH 6.0 and A. bisporus 6.0 was taken as 100%.

Kamal U. Zaidi and Ayesha S. Ali

FIGURE 5: Effect of temperature on the tyrosinase activity of the crude extract prepared from P. ostrea- tus and A. bisporus. Data were obtained as mean value of optical density. The optimum activity of the sample at 35˚C was taken as 100%.

FIGURE 6: Lineweaver-Burk plot of P. ostreatus and A. bisporus tyrosinase. Data were obtained as mean value of 1/ [V] at 475 nm per min (OD 475/min/mg), of triplicate independent tests with different concen- trations of L-DOPA as a substrate.

A similar result has been observed in Lentinula edodes (Kanda et al., 1996) and Lentinula boryana (Faria et al., 2007). On the other hand, optimum pH near neutral has been reported for tyrosinase of Portabella mushrooms (Fan et al., 2004) Agaricus bisporus (Gouzi et al., 2007) and Pycnoporus sanguineus (Halaouli et al., 2005). The tyrosinase isolated sun#ower plant remained fully active between pH4.8 to 7.9 after 20-hour exposure to buffers of different pH at 4˚C. Sang et al. (2005), have reported that in case of recombinant human tyrosinase, the opti- mum pH was 7.5 which as compared to that of presently studied Pleurotus ostreatus, is quite low, being 6.0. The optimum pH of tyrosinase activity of A. bisporus was observed 7.0. While, the optimum pH of tyrosinase activ- ity of P. ostreatus was found 6.0 which is lower than pH value of A. bisporus.

The A. bisporus tyrosinase was active at a wide range of temperature from 30ºC to 65ºC with an optimum at 35ºC (Figure 5), and about 35% of tyrosinase activity was still present at 55ºC, but it lost its activity at 65ºC. Our results were in agreement with a previous study which reported that the optimum temperature for tyrosi- nase activity obtained from Streptomyces sp. was 35ºC. Tyrosinase from Pseudomonas putida and Trichoderma reesei showed maximum activity at 30ºC (Selinheimo et al., 2006; McMahon et al., 2007), and maximum activ- ity of tyrosinase puri!ed from Bacillus megaterium and Lentinula boryana was at 40ºC (Liu et al,. 2005;Faria et al., 2007). Temperature pro!le of P. ostreatus showed that the enzyme had optimum activity at 35ºC (Figure. 5) which is similar to those reported earlier. The optimum temperature for tyrosinase from Pycnoporus sanguineus has been reported to be 25ºC, and 25 to 40ºC for Agari- cus bisporus (Xu et al., 2011). In contrast to the data of the present study, it has been found that the optimum temperature of tyrosinase from Solenum melongena was

high as 65˚C (Lee et al., 1997). The optimum tempera- ture of tyrosinase activity of A. bisporus was observed at 35˚C while, the optimum temperature of tyrosinase activity of P. ostreatus was also found at 35˚C which is similar in temperature to that of A. bisporus

Based on the Lineweaver-Burk analysis, the Km and Vmax values of tyrosinase from A. bisporus tyrosinase was found to be 0.933mM shown in (Figure 6). This result indicates the high af!nity of tyrosinase towards its sub- strate, which might relate to its degree of effectiveness against melanogenesis. Higher Km values 0.9 and 0.85mM for tyrosinase from Pycnoporus sanguineus and Lentinula edodes, respectively, have been reported (Halaouli,et al.,2005;, Kanda et al., 1996). On The other hand, a lower Km value (0.075mM) was obtained for tyrosinase from Bacillus megaterium (Liu et al,. 2005). In P. ostreatus were 0.119 mM and 2.97 mg, respectively (Figure 6).

This indicates the high af!nity of the enzyme to the substrate. Tyrosinase of different sources has different substrate af!nities and probably plays different physiological roles in the enzyme activity. Higher Km values (1.9 mM and 0.9 mM) for tyrosinase from L. boryana and Pycnoporus species, respectively, have been reported (Faria et al., 2007; Halaouli et al., 2005). On the other hand, a lower Km value (0.35 mM) was obtained for tyrosinase from Bacilus megaterium (Shuster et al., 2009). The Km value of tyrosinase of A. bisporus was found to be

0.933 mM and Vmax was 2.34 mM. Although, the Km and Vmax values of tyrosinase of P. ostreatus were 0.119 mM

and 2.17 mM which is lower than A. bisporus tyrosinase.


Tyrosinase constitutes one of the most important groups of commercial enzymes. These enzymes have ample uti- lization in industrial processes, such as pharmaceuticals,

cosmetic and food industries. There are considerable reports indicating the great potential of this enzyme in medicine, agricultural industries, analytical and envi- ronmental purposes. It is also used to produce synthetic melanin which provides protection against radiation and is used as cation exchangers, drug carriers, antioxidants, antiviral agents or immunogens. We conclude that much more research is necessary in these areas if mushroom tyrosinases are to ful!ll their industrial potential. An ini- tial step for preliminary characterization of the enzyme with the comparative evaluation for the biotechnolog- ical potential of the tyrosinase of A. bisporus and P. ostreatus is undertaken. The tyrosinase from A. bisporus and P.ostreatus has economic advantage over the com- mercially synthesized tyrosinases. Thus, the enzyme can be used to produce cross-linked proteins, allowing enzyme biocatalysts to recycle easily and to improve the consistency and texture of proteins. This indicated that puri!ed and characterized mushroom tyrosinase can be a source for therapeutic and industrial use.


The authors are thankful to People’s University, People’s Group, Bhopal, for laboratory facilities and to the Prin- cipal, Sai!a College of Science and Secretary Sai!a Edu- cation Society, Bhopal, for encouragement.


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