Purification, biochemical characterization and performance evaluation of an alkaline serine protease from Aspergillus flavus MTCC 9952 mutant

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• 作者：Santosh Kumar Yadav^a ; ^{santoshbio27@gmail.com" class="auth_mail" title="E-mail the corresponding author} ; Deepali Bisht^b ; Soni Tiwari^c ; Nandan Singh Darmwal^c
• 关键词：Alkaline protease ; Characterization ; Oxidizing agent ; Organic solvent ; Gelatin hydrolysis
• 刊名：Biocatalysis and Agricultural Biotechnology
• 出版年：2015
• 出版时间：October 2015
• 年：2015
• 卷：4
• 期：4
• 页码：667-677
• 全文大小：3654 K

文摘

A ~38 kDa alkaline protease of mutant Aspergillus flavus MTCC 9952 was purified upto homogeneity by 18.5-fold with 21.0% recovery. The protease was optimally active at pH 11.0 and temperature 40 °C. It revealed significant stability in pH range 8.0–11.0 and temperature range 35–50 °C. Casein was the most preferred substrate which displayed K_m=1.95±0.03 mg/ml and V_max=476.2±9.4 µg tyrosine/ml/min. The catalytic activity was enhanced by Fe⁺⁺, Ca⁺⁺ and Ni⁺⁺ while completely inhibited by Cu⁺⁺ and Hg⁺⁺. Phenyl Methyl Sulphonyl Fluoride caused complete inhibition whereas, activity was trivially inhibited by Ethylene Diamine Tetra Acetic Acid and activated by reducing agents beta-mercaptoethanol and di-thiothreitol. The enzyme exhibited excellent stability towards H₂O₂, surfactants and laundry detergents. The purified protease was activated in the presence of benzene (120.1%) and chloroform (122.3%), nevertheless, retained significant stability with Acetone (90.2%), Diethyl ether (93.0%), n-Hexane (92.4%), Octane (99.8%), and Dodecane (95.0%). In application studies, the protease completely removed blood stains from cotton cloths when used with detergents and hydrolyzed gelatin from waste X-ray films. Presumably, this is the first report on alkaline protease from A. flavus, exhibiting organic solvent stability and gelatin hydrolysis from used X-ray films. Conclusively, this protease could potentially be used in detergent formulation, silver recovery from waste X-ray films and peptide synthesis.