Purification and C haractrization of P rotease by Pleurotus eryngii

Publish Year: 1397
نوع سند: مقاله کنفرانسی
زبان: English
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شناسه ملی سند علمی:

CBC15_022

تاریخ نمایه سازی: 29 خرداد 1398

Abstract:

This work focuses to protease production by Pleurotus eryngii using different carbon and nitrogen source with 0.5, and 1.0% concentrations employing submerged fermentation. The highest production rate was recorded with 0.1% sucrose and 1.0% casein as a carbon and nitrogen source.The enzyme was purified 1.377- fold by gel chromatography on Sephadex G-100, and Fraction-1, F2 and F3 were purified 1.379, 1.419, and 1.322- fold on anion exchange CM A-50 and purified enzyme showed high specific activity of 2.420, 2.49 and 2.32 respectively. The molecular weight of purified fraction F1, F2 and F3 were found to be 30, 30 and 35 kDa by SDS-PAGE.The optimal pH and temperature for protease activity was 8 and 65°C, respectively. But from pH 7 to 9 the protease activity remains constant in two fractions after 9 pH activity slowly decline. The temperature profile showed that purified protease activity increases with increase of temperature up to 65°C and activity gradually lost with the increases of temperature due to enzyme denaturation at high temperature. The temperature stability was checked by heating enzyme for 10 minutes prior the addition of substrate and enzyme activity was checked by following procedure of enzyme activity. But still 25 % activity was retained at 80°C for 10 minutes.The Km and Vmax values of purified protease activity of F1, F2 and F3 were observed 0.65mg/ml/0.157 units/min; 0.5mg/ml/0.157units/min and .9mg/ml/1.00units/min respectively. The activation energy of fraction F1, F2 and F3 was calculated 1.1157, 1.1269 and 0.9602 kj/mol respectively. The half-life of Pleurotus eryngii protease was noted 75, 83 and 76°C for fraction F1, F2 and F3 respectively.Although 5mM concentration of AgNO3 reduced the 60% activity of F1, CuSO4 reduced the 64 % activity of F2 and MnSO4 reduced 79% activity of F3. As shown in result CaCL2, Cysteine, MgCl2, ZnCl2, CoCl2, Mercaptoethanol stimulated the protease activity with different rate, which suggest two or more active sites.

Authors

M Umar Dahot

Institute of Biotechnology and Genetic Engineering, University of Sindh, Jamshoro, Sindh, Pakistan

S Bano

Institute of Biotechnology and Genetic Engineering, University of Sindh, Jamshoro, Sindh, Pakistan