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Purification and characterization of melanogenic enzyme tyrosinase from button mushroom.

Zaidi KU, Ali AS, Ali SA - Enzyme Res (2014)

Bottom Line: The purified tyrosinase was optimized and the results revealed that the optimum values are pH 7.0 and temperature 35°C.The highest activity was reported towards its natural substrate, L-DOPA, with an apparent Km value of 0.933 mM.This indicated that tyrosinase purified from Agaricus bisporus is a potential source for medical applications.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biotechnology Laboratory, Centre for Scientific Research & Development, People's University, Bhopal 462010, India.

ABSTRACT
Melanogenesis is a biosynthetic pathway for the formation of the pigment melanin in human skin. A key enzyme, tyrosinase, catalyzes the first and only rate-limiting steps in melanogenesis. Since the discovery of its melanogenic properties, tyrosinase has been in prime focus and microbial sources of the enzyme are sought. Agaricus bisporus widely known as the common edible mushroom, it's taking place in high amounts of proteins, enzyme, carbohydrates, fibers, and low fat contents are frequently cited in the literature in relation to their nutritional value. In the present study tyrosinase from Agaricus bisporus was purified by ammonium sulphate precipitation, dialysis followed by gel filtration chromatography on Sephadex G-100, and ion exchange chromatography on DEAE-Cellulose; the enzyme was purified, 16.36-fold to give 26.6% yield on total activity in the crude extract and final specific activity of 52.19 U/mg. The SDS-PAGE electrophoresis showed a migrating protein band molecular weight of 95 kDa. The purified tyrosinase was optimized and the results revealed that the optimum values are pH 7.0 and temperature 35°C. The highest activity was reported towards its natural substrate, L-DOPA, with an apparent Km value of 0.933 mM. This indicated that tyrosinase purified from Agaricus bisporus is a potential source for medical applications.

No MeSH data available.


Effect of pH on the tyrosinase activity of the crude extract of A. bisporus. Data were obtained as mean value of optical density. Assays were done at 35°C and the activity of the sample was incubated on 100 mM acetate buffer at 4.0-5.0 pH, 100 mM phosphate buffer at 6.0–8.0 pH, and 100 mM Tris-HCl buffer at 9.0-10 pH. The optimum activity of the sample at pH 6.0 was taken as 100%.
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fig5: Effect of pH on the tyrosinase activity of the crude extract of A. bisporus. Data were obtained as mean value of optical density. Assays were done at 35°C and the activity of the sample was incubated on 100 mM acetate buffer at 4.0-5.0 pH, 100 mM phosphate buffer at 6.0–8.0 pH, and 100 mM Tris-HCl buffer at 9.0-10 pH. The optimum activity of the sample at pH 6.0 was taken as 100%.

Mentions: 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 [27, 33]. Results (Figure 5) 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. [31] 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 [27]. 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.


Purification and characterization of melanogenic enzyme tyrosinase from button mushroom.

Zaidi KU, Ali AS, Ali SA - Enzyme Res (2014)

Effect of pH on the tyrosinase activity of the crude extract of A. bisporus. Data were obtained as mean value of optical density. Assays were done at 35°C and the activity of the sample was incubated on 100 mM acetate buffer at 4.0-5.0 pH, 100 mM phosphate buffer at 6.0–8.0 pH, and 100 mM Tris-HCl buffer at 9.0-10 pH. The optimum activity of the sample at pH 6.0 was taken as 100%.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4150416&req=5

fig5: Effect of pH on the tyrosinase activity of the crude extract of A. bisporus. Data were obtained as mean value of optical density. Assays were done at 35°C and the activity of the sample was incubated on 100 mM acetate buffer at 4.0-5.0 pH, 100 mM phosphate buffer at 6.0–8.0 pH, and 100 mM Tris-HCl buffer at 9.0-10 pH. The optimum activity of the sample at pH 6.0 was taken as 100%.
Mentions: 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 [27, 33]. Results (Figure 5) 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. [31] 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 [27]. 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.

Bottom Line: The purified tyrosinase was optimized and the results revealed that the optimum values are pH 7.0 and temperature 35°C.The highest activity was reported towards its natural substrate, L-DOPA, with an apparent Km value of 0.933 mM.This indicated that tyrosinase purified from Agaricus bisporus is a potential source for medical applications.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biotechnology Laboratory, Centre for Scientific Research & Development, People's University, Bhopal 462010, India.

ABSTRACT
Melanogenesis is a biosynthetic pathway for the formation of the pigment melanin in human skin. A key enzyme, tyrosinase, catalyzes the first and only rate-limiting steps in melanogenesis. Since the discovery of its melanogenic properties, tyrosinase has been in prime focus and microbial sources of the enzyme are sought. Agaricus bisporus widely known as the common edible mushroom, it's taking place in high amounts of proteins, enzyme, carbohydrates, fibers, and low fat contents are frequently cited in the literature in relation to their nutritional value. In the present study tyrosinase from Agaricus bisporus was purified by ammonium sulphate precipitation, dialysis followed by gel filtration chromatography on Sephadex G-100, and ion exchange chromatography on DEAE-Cellulose; the enzyme was purified, 16.36-fold to give 26.6% yield on total activity in the crude extract and final specific activity of 52.19 U/mg. The SDS-PAGE electrophoresis showed a migrating protein band molecular weight of 95 kDa. The purified tyrosinase was optimized and the results revealed that the optimum values are pH 7.0 and temperature 35°C. The highest activity was reported towards its natural substrate, L-DOPA, with an apparent Km value of 0.933 mM. This indicated that tyrosinase purified from Agaricus bisporus is a potential source for medical applications.

No MeSH data available.