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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.


Elution profile on DEAE-Cellulose column chromatography. An aliquot of each fraction was assayed for protein content and tyrosinase activity. A linear gradient of NaCl was 0–100 mM.
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fig1: Elution profile on DEAE-Cellulose column chromatography. An aliquot of each fraction was assayed for protein content and tyrosinase activity. A linear gradient of NaCl was 0–100 mM.

Mentions: Briefly, crude extracts were prepared by homogenization in 100 mM potassium phosphate buffer (pH 5.8) containing 1 mM EDTA. After centrifugation, the supernatant was applied to ammonium sulfate precipitation. Partial purification of tyrosinase using ammonium sulfate precipitation showed that the best fraction was 70% with respect to the crude enzyme and other fractions. It gave the maximum values of total activity, specific activity, and yield of the tyrosinase enzyme which reached 11.09 U/mg, 83.5%, respectively. The -fold purification of the purified enzyme was 3.47 when 70% ammonium sulfate was used. The previous findings were identical to that reported by Lee et al. [19], who found that 70% ammonium sulfate was the best fraction which gave the highest yield of tyrosinase activity from Solanum melongena. The dialyzed ammonium sulfate precipitate that was applied to Sephadex G-100 gel filtration column chromatography showed major peaks of tyrosinase activity which were observed in active fractions and resulted in 9.22-fold purification with a final specific activity of 29.42 U/mg. The overall recovery of the purification was 35.7% (Table 1). This active fraction was applied to a DEAE-Cellulose column and eluted with a stepwise-increasing NaCl gradient. The enzyme was eluted at 0–100 mM NaCl (Figure 1). The eluted active fractions rechromatographed on the same column with a linear gradient of potassium phosphate buffer (0–100 mM) were passed through the column (Figure 2). This two-step purification scheme, ion exchange chromatography, resulted in a partially purified tyrosinase preparation, obtained by pooling fractions 25, 26, 27, and 28 and the enzyme was purified by about 16.36-fold purification with a final specific activity of 52.19 U/mg. The overall recovery of the purification was 26.6%. Horowitz et al. [20] reported that tyrosinase that is produced in the fruiting body can be recuperated and purified by homogenizing in a blender and then passed through a French press followed by acetone or ammonium sulfate precipitation [21]. The resuspended precipitate was further purified by one or more chromatography columns. The most commonly used columns are hydroxylapatite [22], DEAE-Cellulose [23] or DEAE Sepharose [24], various other immunoaffinity resins [25], and Sephadex size exclusion gel [21].


Purification and characterization of melanogenic enzyme tyrosinase from button mushroom.

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

Elution profile on DEAE-Cellulose column chromatography. An aliquot of each fraction was assayed for protein content and tyrosinase activity. A linear gradient of NaCl was 0–100 mM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4150416&req=5

fig1: Elution profile on DEAE-Cellulose column chromatography. An aliquot of each fraction was assayed for protein content and tyrosinase activity. A linear gradient of NaCl was 0–100 mM.
Mentions: Briefly, crude extracts were prepared by homogenization in 100 mM potassium phosphate buffer (pH 5.8) containing 1 mM EDTA. After centrifugation, the supernatant was applied to ammonium sulfate precipitation. Partial purification of tyrosinase using ammonium sulfate precipitation showed that the best fraction was 70% with respect to the crude enzyme and other fractions. It gave the maximum values of total activity, specific activity, and yield of the tyrosinase enzyme which reached 11.09 U/mg, 83.5%, respectively. The -fold purification of the purified enzyme was 3.47 when 70% ammonium sulfate was used. The previous findings were identical to that reported by Lee et al. [19], who found that 70% ammonium sulfate was the best fraction which gave the highest yield of tyrosinase activity from Solanum melongena. The dialyzed ammonium sulfate precipitate that was applied to Sephadex G-100 gel filtration column chromatography showed major peaks of tyrosinase activity which were observed in active fractions and resulted in 9.22-fold purification with a final specific activity of 29.42 U/mg. The overall recovery of the purification was 35.7% (Table 1). This active fraction was applied to a DEAE-Cellulose column and eluted with a stepwise-increasing NaCl gradient. The enzyme was eluted at 0–100 mM NaCl (Figure 1). The eluted active fractions rechromatographed on the same column with a linear gradient of potassium phosphate buffer (0–100 mM) were passed through the column (Figure 2). This two-step purification scheme, ion exchange chromatography, resulted in a partially purified tyrosinase preparation, obtained by pooling fractions 25, 26, 27, and 28 and the enzyme was purified by about 16.36-fold purification with a final specific activity of 52.19 U/mg. The overall recovery of the purification was 26.6%. Horowitz et al. [20] reported that tyrosinase that is produced in the fruiting body can be recuperated and purified by homogenizing in a blender and then passed through a French press followed by acetone or ammonium sulfate precipitation [21]. The resuspended precipitate was further purified by one or more chromatography columns. The most commonly used columns are hydroxylapatite [22], DEAE-Cellulose [23] or DEAE Sepharose [24], various other immunoaffinity resins [25], and Sephadex size exclusion gel [21].

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.