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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.


Polyacrylamide gel electrophoresis of tyrosinase from A. bisporus, lane A, standard protein of different molecular weight; lane B, crude extract; lane C, ammonium sulfate fraction; lane D, dialysis; lane E, Sephadex G-100 gel filtration fraction; and lane F, DEAE-Cellulose fraction of tyrosinase ~95 kDa.
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fig3: Polyacrylamide gel electrophoresis of tyrosinase from A. bisporus, lane A, standard protein of different molecular weight; lane B, crude extract; lane C, ammonium sulfate fraction; lane D, dialysis; lane E, Sephadex G-100 gel filtration fraction; and lane F, DEAE-Cellulose fraction of tyrosinase ~95 kDa.

Mentions: SDS-PAGE of the enzyme preparation from different purification steps showed that the resolved electrophoretic bands were progressively improved from the crude extract to the final 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 95 kDa (Figure 3). In this respect, tyrosinase purified from Aspergillus oryzae, Trichoderma reesei, and Aspergillus nidulans was with smaller molecular weight in the range of 67, 43.2, and 50.48 kDa [26–28]. Kanda et al. [29] obtained two activity peaks after ion exchange chromatography of an extract from Lentinula edodes. When the fractions corresponding 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 fractions gave bands at 15, 49, and 54 kDa for one fraction and 15, 50, and 55 kDa for the other purified tyrosinase from Lentinula edodes [29] and exhibited a molecular weight of 105 kDa.


Purification and characterization of melanogenic enzyme tyrosinase from button mushroom.

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

Polyacrylamide gel electrophoresis of tyrosinase from A. bisporus, lane A, standard protein of different molecular weight; lane B, crude extract; lane C, ammonium sulfate fraction; lane D, dialysis; lane E, Sephadex G-100 gel filtration fraction; and lane F, DEAE-Cellulose fraction of tyrosinase ~95 kDa.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Polyacrylamide gel electrophoresis of tyrosinase from A. bisporus, lane A, standard protein of different molecular weight; lane B, crude extract; lane C, ammonium sulfate fraction; lane D, dialysis; lane E, Sephadex G-100 gel filtration fraction; and lane F, DEAE-Cellulose fraction of tyrosinase ~95 kDa.
Mentions: SDS-PAGE of the enzyme preparation from different purification steps showed that the resolved electrophoretic bands were progressively improved from the crude extract to the final 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 95 kDa (Figure 3). In this respect, tyrosinase purified from Aspergillus oryzae, Trichoderma reesei, and Aspergillus nidulans was with smaller molecular weight in the range of 67, 43.2, and 50.48 kDa [26–28]. Kanda et al. [29] obtained two activity peaks after ion exchange chromatography of an extract from Lentinula edodes. When the fractions corresponding 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 fractions gave bands at 15, 49, and 54 kDa for one fraction and 15, 50, and 55 kDa for the other purified tyrosinase from Lentinula edodes [29] and exhibited a molecular weight of 105 kDa.

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.