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Characterization of Thermostable Serine Alkaline Protease from an Alkaliphilic Strain Bacillus pumilus MCAS8 and Its Applications

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Abstract

This study describes the characterization and optimization of medium components for an extracellular detergent, surfactant, organic solvent and thermostable serine alkaline protease produced by alkaliphilic Bacillus pumilus MCAS8 strain isolated from Pulicat lake sediments, Tamil Nadu, India. The strain yielded maximum protease (2,214 U/ml) under optimized conditions: carbon source, citric acid—1.5 % (w/w); inducer, soyabean meal—2 % (w/w); pH 11.0; shaking condition 37 °C for 48 h. The enzyme had pH and temperature optima of 9.0 and 60 °C, respectively. The enzyme displayed the molecular mass of 36 kDa in sodium dodecyl sulphate–polyacrylamide gel electrophoresis study and exhibited activity at a wide range of pH (6.0–11.0) and thermostability (20–70 °C). More than 70 % residual activity was observed when the enzyme was incubated with dithiothreitol, ethylenediaminetetraacetic acid, ethylene glycol tetraacetic acid and H2O2 for 30 min. The protease activity was also enhanced by divalent cations such as Ba2+, Ca2+ and Mg2+ and was strongly inhibited by Fe2+, Zn2+, Sr2+, Hg2+ and urea. The enzyme retained more than 50 % of its initial activity after pre-incubation for 1 h in the presence of 5 % (v/v) organic solvents such as dimethyl sulphoxide and acetone. The protease could hydrolyse various native proteinaceous substrates (1 % w/v) such as bovine serum albumin, casein, skim milk, gelatine, azocasein and haemoglobin. Wash performance analysis of enzyme revealed that it could effectively remove blood stains from the cotton fabric, thus making it suitable to use as an effective detergent additive. The protease enzyme also exhibited promising result in the dehairing of goat skin. The potency of the eco-friendly enzyme without using any chemicals against washing and dehairing showed that the enzyme could be used for various industrial applications.

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Acknowledgments

The authors thank the Council for Scientific and Industrial Research, Government of India, New Delhi, India for financial assistance through a research project under Coastal Hazard Preparedness [No. 23 (0010)/07/EMR-11]. The authors also thank Ms. S. Devilakshmi, research scholar, Department of Biotechnology, IIT Madras, Chennai for her assistance in enzyme purification and zymogram analysis.

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  1. Shri AMM Murugappa Chettiar Research Center, Taramani, Chennai, 600113, India

    Renganathan Jayakumar, Shanmugam Jayashree, Balumuri Annapurna & Sundaram Seshadri

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  1. Renganathan Jayakumar
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Correspondence to Sundaram Seshadri.

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Jayakumar, R., Jayashree, S., Annapurna, B. et al. Characterization of Thermostable Serine Alkaline Protease from an Alkaliphilic Strain Bacillus pumilus MCAS8 and Its Applications. Appl Biochem Biotechnol 168, 1849–1866 (2012). https://doi.org/10.1007/s12010-012-9902-6

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