Screening, Production and characterization of lacasse from fusant strain of
The current work emphasizes on the screening, production and characterization of laccase from improved strain of Pleurotus species. Temperature stabilities of laccases were found to be 70°C. The optimum pH of the laccase differed with the substrate used. The optimum pH for the guaiacol was 7.0. The effect of different metal ions on the activity of laccase enzyme was investigated. Results showed that all the metal ions used exhibited inhibition effects to the enzyme activity but with different degrees. FeSO4 showed complete inhibition effect on laccase enzyme. The metal ions KCl and NaCl have less effect on the enzyme activity where the enzyme retained about 71% of its initial activity. Effect of a range of potent laccase inhibitors on the enzyme activity was tested. The oxidase inhibitors sodium azide and sodium cyanide caused about 77.77 and 51.48% inhibition of laccase activity respectively. However the enzyme retained 69.04% of its activity in the presence of 1 mM SDS. Other inhibitors, EDTA and aniline have low effect on laccase activity. The effect of enzyme concentration on enzyme activity was carried out under standard conditions with varying amounts of enzyme (mg protein) added. The results showed that, the maximum laccase activity was obtained with 0.0036 mg protein of the purified enzyme per reaction mixture. The effect of substrate (guaiacol) concentration on the activity of the purified laccase enzyme was studied. Results indicated that the activity increased linearly up on increasing the guaiacol concentration to reach maximum at 40 mM and higher substrate concentrations than 40 mM resulted in a constant enzyme activity. The kinetic parameters (Km and Vmax values) of the purified laccase enzyme was determined by using guaicol as a substrate and calculated from Lineweaver and Burk plots. The results indicated that the Km value was 71.43mM and Vmax value was 142.86U/ml protein. The laccase from Pleurotus species was purified using DEAE-Cellulose and eluted with NaCl showed the highest laccase activity. The molecular mass of purified laccases was found to be approximately 43 kDa.
Key Words: Laccase, Purification, Pleurotus, DEAE- Cellulose, Kinetic parameters
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